Linda Chaabane

Dose-Related Effects of Repeated ETC-216 (Recombinant Apolipoprotein A-IMilano/1-Palmitoyl-2-Oleoyl Phosphatidylcholine Complexes) Administrations on Rabbit Lipid-Rich Soft Plaques. In Vivo Assessment by Intravascular Ultrasound and Magnetic Resonance Imaging

OBJECTIVES This study sought to evaluate in vivo the minimal dose of apolipoprotein (apo) A-I(Milano) phospholipid complex (recombinant apoA-I(Milano) and 1-palmitoyl-2-oleoyl phosphatidylcholine complexes [ETC-216]) able to induce atherosclerosis regression in a rabbit model of lipid-rich plaques. BACKGROUND A single high dose of recombinant apoA-I(Milano) has promoted atherosclerosis regression in animal models. More recently, regression of atherosclerosis was achieved in coronary patients by repeated infusions of ETC-216. METHODS Thirty-six rabbits underwent perivascular injury at both carotid arteries, followed by a 1.5% cholesterol diet. After 90 days, rabbits were randomly divided into 6 groups and treated 5 times with vehicle or ETC-216 at 5, 10, 20, 40, or 150 mg/kg dose every 4 days. Carotid plaque changes were evaluated in vivo by intravascular ultrasound (IVUS) and magnetic resonance imaging (MRI), performed before and at the end of treatments. Magnetic resonance imaging scans were also recorded after administration of the second dose for rabbits infused with vehicle 40 or 150 mg/kg. RESULTS Atheroma volume in vehicle-treated rabbits increased dramatically between the first and the second IVUS analyses (+26.53%), whereas in ETC-216-treated animals, a reduced progression at the lower doses and a significant regression at the higher doses, up to -6.83%, was detected. Results obtained by MRI analysis correlated significantly with those at IVUS (r = 0.706; p < 0.0001). The MRI evaluations after the second infusion established that a significant regression was achieved with only 2 administrations of the highest dose. CONCLUSIONS These results confirm the efficacy of ETC-216 for atherosclerosis treatment and provide guidance for dose selection and frequency to obtain a significant reduction of plaque volume.

Fingolimod May Support Neuroprotection via Blockade of Astrocyte Nitric Oxide

Although astrocytes participate in glial scar formation and tissue repair, dysregulation of the NFκB pathway and of nitric oxide (NO) production in these glia cells contributes to neuroinflammation and neurodegeneration. Here we investigated the role of the crosstalk between sphingosine‐1‐phosphate (S1P) and cytokine signaling cascades in astrocyte activation and inflammation‐mediated neurodegeneration, and addressed the effects of fingolimod on astrocyte–neuron interaction and NO synthesis in vivo.

Macrophages and liposomes in inflammatory disease : Friends or foes?

Liposome-encapsulated corticosteroids have shown to exert strong beneficial effects in inflammatory diseases, such as arthritis and cancer. To extend the clinical applicability of these potent nanomedicines, the therapeutic effect of dexamethasone phosphate loaded long-circulating liposomes (LCL-DXP) was evaluated in animal models of multiple sclerosis (MS) and Crohns disease (CD). In mice with experimental autoimmune encephalitis (EAE), a model for MS, treatment with LCL-DXP, but not free DXP, resulted in a decrease in disease activity when compared to PBS treated mice. In contrast, in mice with chronic DSS-induced colitis, a model for CD, treatment with LCL-DXP did not induce an improvement, but in fact worsened the fecal blood loss after treatment, indicating an aggravation of the disease. It is hypothesized that modulation of macrophage polarization towards a M2 phenotype underlies the efficacy of corticosteroid-based drug delivery systems, which is supported by the presented data. On the one hand, M1 polarized macrophages are part of the pathogenesis of MS; the modulation to M2-polarization by LCL-DXP is therefore beneficial. On the other hand, M1-polarized intestinal macrophages fulfill a protective and inflammation-suppressing role in intestinal homeostasis; changing their phenotype to M2 causes reduced protection to invading microorganisms, leading to a more severe intestinal inflammation. These findings therefore indicate that the interplay between the specific phenotype of macrophages and the specific inflammatory context of the inflammatory disease in question may be an important determining factor in the therapeutic applicability of liposomal corticosteroids in inflammatory disease.

Contrast enhancement in atherosclerosis development in a mouse model: in vivo results at 2 Tesla

To develop an MRI method for the evaluation of contrast enhancement in early atherosclerotic plaque development in the abdominal aorta of a mouse model. Male apoE−/− mice from three groups, respectively 4 (n = 6), 8 (n = 11) and 16 (n = 4) weeks were included. Axial T1 spin echo images of the abdominal aorta were obtained above and below the renal arteries (90 μm spatial resolution) before and over 1 h after the injection of a macromolecular contrast agent. Signal enhancement was measured in the vessel wall and compared to histological features. Maximal arterial wall signal enhancement was obtained from 16 to 32 min post injection. During this time, the signal-to-noise ratio increased by a factor up to 1.7 in 16 week mice and 2.7 and 2.4 in 8 and 4 weeks mice, respectively. The enhancement of the arterial wall appeared less pronounced in the oldest mice, 16 weeks old, exhibiting more advanced lesions. Using a macromolecular gadolinium agent, contrast uptake in atherogenesis varies with lesion stage and may be related to vessel-wall permeability. Dynamic contrast-enhanced MRI may be useful to evaluate the atherosclerotic plaque activity in mice.

Evidence for in vivo macrophage mediated tumor uptake of paramagnetic/fluorescent liposomes

Dysprosium (Dy)‐loaded liposomes act as excellent T2‐susceptibility agents at high magnetic field strength. The R2‐enhancement increases with the size of the liposomes and the concentration of entrapped paramagnetic metal complexes. Neuro‐2a tumor cells are readily labeled when Dy‐loaded liposomes, suitably functionalized with glutamine residues (Gln), are added to the culture medium as glutamine receptors are highly expressed in such proliferating tumor cells. By using fluorescent liposomes doped with fluorescent dyes (either incorporated in the membrane or included in the inner cavity), confocal microscopy experiments showed that targeted liposomes are taken up much more avidly than non‐targeted vesicles. In vivo studies showed that glutamine‐functionalized and non‐functionalized liposomes accumulate in the tumor region to a similar extent. Confocal images of the excised tumor showed extensive co‐localization of liposomes and macrophages in both cases. It is suggested that the loss of tumor specificity, shown by Gln‐functionalized liposomes in vivo, has to be associated with the efficient removal of liposomes operated by the RES (reticulo endoplasmatic system) or tumor associated macrophages. Copyright

Biomimetic MRI contrast agent for imaging of inflammation in atherosclerotic plaque of ApoE-/- mice: a pilot study.

Objective:Atherosclerosis involves an inflammatory process characterized by cellular and molecular responses. A slow-clearance blood-pool paramagnetic agent (CMD-A2-Gd-DOTA: P717) chemically modified to create a functionalized product (F-P717) for targeting inflammation in vessel walls was evaluated in vivo in mice. Methods and Results:Carboxylate and sulfate groups were grafted onto the macromolecular paramagnetic Gd-DOTA-dextran backbone. Products were also fluorescently labeled with rhodamine isothiocyanate. Pre- and postcontrast MRI was performed on a 2-Tesla magnet in ApoE−/− and control C57BL/6 mice after P717 or F-P717 injection at a dose of 60 &mgr;mol Gd/kg. Axial T1-weighted images of the abdominal aorta were obtained using a 2D multislice spin-echo sequence. F-P717 significantly enhanced the magnetic resonance imaging (MRI) signal in the abdominal aortic wall of ApoE−/− mice (>50% signal-to-noise ratio increase between 10 and 30 minutes), but not of control mice. P717 produced only moderate (<20%) MRI signal enhancement within the same time frame. The MRI data were correlated to histopathology. Immunofluorescence in ApoE−/− mice colocalized F-P717 but not P717 with the inflammatory area revealed by P-selectin labeling. Conclusion:This study demonstrates the efficacy of F-P717 as a new molecular imaging agent for noninvasive in vivo MRI location of inflammatory vascular tree lesions in ApoE−/− mice.

Quantification of Multicontrast Vascular MR Images with NLSnake, an Active Contour Model: In Vitro Validation and In Vivo Evaluation

Vessel‐wall measurements from multicontrast MRI provide information on plaque structure and evolution. This requires the extraction of numerous contours. In this work a contour‐extraction method is proposed that uses an active contour model (NLSnake) adapted for a wide range of MR vascular images. This new method employs length normalization for the purpose of deformation computation and offers the advantages of simplified parameter tuning, fast convergence, and minimal user interaction. The model can be initialized far from the boundaries of the region to be segmented, even by only one pixel. The accuracy and reproducibility of NLSnake endoluminal contours were assessed on vascular phantom MR angiography (MRA) and high‐resolution in vitro MR images of rabbit aorta. An in vivo evaluation was performed on rabbit and clinical data for both internal and external vessel‐wall contours. In phantoms with 95% stenoses, NLSnake measured 94.3% ± 3.8%, and the accuracy was even better for milder stenoses. In the images of rabbit aorta, variability between NLSnake and experts was less than interobserver variability, while the maximum intravariability of NLSnake was equal to 1.25%. In conclusion, the NLSnake technique successfully quantified the vessel lumen in multicontrast MR images using constant parameters. Magn Reson Med 51:370–379, 2004.

A R2p /R1p ratiometric procedure to assess matrix metalloproteinase-2 activity by magnetic resonance imaging.

The approach to molecular imaging of enzymes by MRI typically relies upon imaging probes composed of an enzymecleavable moiety conjugated with a paramagnetic imaging reporter, such as a Gd chelate. Upon enzymatic processing, the probe is transformed into a fragment with an altered relaxivity, leading to a different capability to enhance contrast in MR images with respect to the parent species. Ideally, the unprocessed (intact) form of the probe should be completely silent while the processed (cleaved) form should have a high relaxivity (that is, high contrast enhancement). In such a way the appearance of contrast within images can be unambiguously attributed to the result of enzymatic activity and not to dynamic changes of tissue probe concentration. However, gadolinium-based agents as enzyme responsive agents are never completely silent and both forms (unprocessed and processed) contribute to the overall contrast enhancement as a function of their respective relaxivities and tissue local concentrations. Exact knowledge of the total concentration of Gd is essential to translate image contrast enhancement into the molar ratio of unprocessed versus processed forms, and thus into true enzyme activity maps. A viable solution to the concentration problem can be provided by the R2p/R1p ratiometric approach, which is based on the measurement of the ratio between the transverse and longitudinal paramagnetic contributions to the water proton relaxation rate; that is, R2p and R1p (with Ri = 1/Ti, i = 1,2). Although ratiometric approaches are well-established for CEST-MRI contrast agents, there are only a couple of examples of application to Gd based relaxation agents, namely for pH and temperature imaging. 5] Let us consider two Gd complexes, GdL and GdF, each characterized by its own transverse and longitudinal millimolar relaxivity (r2 and r1 terms respectively, in units of mm 1 s ). These species form the ratiometric pair, and the total paramagnetic relaxation enhancement of a mixture of the two can be expressed as:

Novel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activity

Transglutaminases, including factor XIII and tissue transglutaminase, participate in multiple extracellular processes associated with remodeling of the extracellular matrix during wound repair, blood clotting, tumor progression and fibrosis of ischemic injuries. The aim of this work was to evaluate a novel substrate analog for transglutaminase optimized by molecular modeling calculations (DCCP16), which can serve for molecular imaging of transglutaminase activity by magnetic resonance imaging and by near-infrared imaging. Experimental data showed covalent binding of Gd-DCCP16 and DCCP16-IRIS Blue to human clots, to basement membrane components and to casein in purified systems as well as in three-dimensional multicellular spheroids. In vivo, DCCP16 showed enhancement with a prolonged retention in clots and tumors, demonstrating the ability to detect both factor XIII and tissue transglutaminase mediated covalent binding of the contrast material.

Novel Gd(III)-based probes for MR molecular imaging of matrix metalloproteinases

Two novel Gd-based contrast agents (CAs) for the molecular imaging of matrix metalloproteinases (MMPs) were synthetized and characterized in vitro and in vivo. These probes were based on the PLG*LWAR peptide sequence, known to be hydrolyzed between Gly and Leu by a broad panel of MMPs. A Gd-DOTA chelate was conjugated to the N-terminal position through an amide bond, either directly to proline (compd Gd-K11) or through a hydrophilic spacer (compd Gd-K11N). Both CA were made strongly amphiphilic by conjugating an alkyl chain at the C-terminus of the peptide sequence. Gd-K11 and Gd-K11N have a good affinity for β-cyclodextrins (K(D) 310 and 670 µ m respectively) and for serum albumin (K(D) 350 and 90 µ m respectively), and can be efficiently cleaved in vitro at the expected site by MMP-2 and MMP-12. Upon MMP-dependent cleavage, the CAs lose the C-terminal tetrapeptide and the alkyl chain, thus undergoing to an amphiphilic-to-hydrophilic transformation that is expected to alter tissue pharmacokinetics. To prove this, Gd-K11 was systemically administered to mice bearing a subcutaneous B16.F10 melanoma, either pre-treated or not with the broad spectrum MMP inhibitor GM6001 (Ilomastat). The washout of the Gd-contrast enhancement in MR images was significantly faster for untreated subjects (displaying MMP activity) with respect to treated ones (MMP activity inhibited). The washout kinetics of Gd-contrast enhancement from the tumor microenvironment could be then interpreted in terms of the local activity of MMPs.