Magnetic Resonance Imaging Reveals Distinct Roles for Tissue Transglutaminase and Factor XIII in Maternal Angiogenesis During Early Mouse Pregnancy.

OBJECTIVE: The early embryo implantation is characterized by enhanced uterine vascular permeability at the site of blastocyst attachment, followed by extracellular-matrix remodeling and angiogenesis. Two TG (transglutaminase) isoenzymes, TG2 (tissue TG) and FXIII (factor XIII), catalyze covalent cross-linking of the extracellular-matrix. However, their specific role during embryo implantation is not fully understood. Approach and Results: For mapping the distribution as well as the enzymatic activities of TG2 and FXIII towards blood-borne and resident extracellular-matrix substrates, we synthetized selective and specific low molecular weight substrate analogs for each of the isoenzymes. The implantation sites were challenged by genetically modifying the trophoblast cells in the outer layer of blastocysts, to either overexpress or deplete TG2 or FXIII, and the angiogenic response was studied by dynamic contrast-enhanced-magnetic resonance imaging. Dynamic contrast-enhanced-magnetic resonance imaging revealed a decrease in the permeability of decidual vasculature surrounding embryos in which FXIII were overexpressed in trophoblast cell. Reduction in decidual blood volume fraction was demonstrated when either FXIII or TG2 were overexpressed in embryonic trophoblast cell and was elevated when trophoblast cell was depleted of FXIII. These results were corroborated by histological analysis. CONCLUSIONS: In this study, we report on the isoenzyme-specific roles of TG2 and FXIII during the early days of mouse pregnancy and further reveal their involvement in decidual angiogenesis. Our results reveal an important magnetic resonance imaging-detectable function of embryo-derived TG2 and FXIII on regulating maternal angiogenesis during embryo implantation in mice.Visual Overview: An online visual overview is available for this article.

An efficient MRI agent targeting extracellular markers in prostate adenocarcinoma.

PURPOSE: Prostate cancer (PCa) is the most widespread tumor affecting males in Western countries. We propose a novel MRI molecular tetrameric probe based on the heptadentate gadolinium (Gd)-AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) that is able to in vivo detect PCa through the recognition of the fibrin-fibronectin (FB-FN) complex. METHODS: The peptide CREKA (Cys-Arg-Glu-Lys-Ala), targeting the FB-FN complex in the reactive stroma of the tumor, was synthesized by solid phase peptide synthesis (SPPS) and conjugated to the tetramer dL-(Gd-AAZTA)4 . The resulting probe was characterized by 1 H relaxometry, tested in vitro on FB clots and in vivo on an orthotopic mouse model of PCa. RESULTS: CREKA-dL-(Gd-AAZTA)4 showed a remarkable relaxivity of 18.2 m MGd-1s-1 (0.47 T, 25°C) because of the presence of 2 water molecules (q = 2) in the inner coordination sphere of each Gd3+ ion, whose rotational motion (τR ) is lengthened as the result of the relatively high molecular weight. The probe displayed a detectable affinity for plasma-derived FB clots. On intravenous injection of the probe in an orthotopic mouse model of PCa, a significant increase in the prostate T1 contrast (~40%) was observed. The MRI signal appears statistically higher either with respect to the one observed for the control probes and to the one detected when CREKA-dL-(Gd-AAZTA)4 was administered to healthy animals. CONCLUSIONS: This study demonstrated the ability of the CREKA-dL-(Gd-AAZTA)4 probe to specifically localize in prostate tumor after injection. The high relaxivity of the probe allows the reduction of the injected dose to 20 µmolGd /kg, yielding a good in vivo contrast enhancement in the region of prostate tumor.

Synthesis of High Relaxivity Gadolinium AAZTA Tetramers as Building Blocks for Bioconjugation.

Molecular imaging requires the specific accumulation of contrast agents at the target. To exploit the superb resolution of MRI for applications in molecular imaging, gadolinium chelates, as the MRI contrast agents (CA), have to be conjugated to a specific vector able to recognize the epitope of interest. Several Gd(III)-chelates can be chemically linked to the same binding vector in order to deliver multiple copies of the CA (multimers) in a single targeting event thus increasing the sensitivity of the molecular probe. Herein three novel bifunctional agents, carrying one functional group for the bioconjugation to targeting vectors and four Gd(III)-AAZTA chelate functions for MRI contrast enhancement (AAZTA = 6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid), are reported. The relaxivity in the tetrameric derivatives is 16.4 ± 0.2 mMGd-1 s-1 at 21.5 MHz and 25 °C, being 2.4-fold higher than that of parent, monomeric Gd(III)-AAZTA. These compounds can be used as versatile building blocks to insert preformed, high relaxivity, and high density Gd-centers to biological targeting vectors. As an example, we describe the use of these bifunctional Gd(III)-chelates to label a fibrin-targeting peptide.

MRI visualization of neuroinflammation using VCAM-1 targeted paramagnetic micelles.

The detection of neuroinflammatory processes using innovative and non-invasive imaging techniques is of great help to deeply investigate the onset and progression of neurodegenerative diseases. Since Vascular Cell Adhesion Molecule (VCAM-1) is over expressed at the blood brain barrier in the event of neuroinflammation, the goal of this work was the testing of MRI detectable micelles targeted towards VCAM-1 to visualize inflamed regions in a mouse model of acute neuroinflammation. The developed probe allowed for the early detection of the disease, with higher T1 signal enhancement and more precise localization in comparison to untargeted micelles or to the clinically approved contrast agent MultiHance. Moreover, the relatively long blood half-life of the nanosystem (ca. 6.3 h) guaranteed a good accumulation in the inflamed regions, paving the way to future diagnostic/theranostic applications, implying the loading of neuroprotective or even anti-cancer drugs inside the core of the micelles.

Paramagnetic Phospholipid-Based Micelles Targeting VCAM-1 Receptors for MRI Visualization of Inflammation.

Inflammation is signaled by the overexpression of epitopes on the vascular endothelium that primarily aim at recruiting immune cells into the inflamed area. The intravascular localization of these biomarkers makes them suitable targets for the MRI visualization of inflammation. Phospholipid-based nanosystems appear excellent candidates in virtue of their good biocompatibility, ability to deliver a high number of imaging units at the target site, and for the easy functionalization with targeting vectors. In this work, phospholipid-based micelles (hydrodynamic diameter of 20 nm) loaded with the amphiphilic Gd(III)-complex Gd-DOTAMA(C18)2 were vectorized with a small peptide able to specifically bind VCAM-1 receptors. The micelles displayed a high longitudinal relaxivity (36.4 s(-1)mmolGd(-1) at 25 °C and 0.7 T). A (1)H- and (17)O-water relaxometry study indicated that the paramagnetic complex embedded in the nanoparticles adopted two isomeric conformations, likely reflecting the well-known square antiprismatic (SAP) and twisted square antiprismatic (TSAP) configurations typically observed in DOTA-like lanthanide complexes. Interestingly, the TSAP structure, showing a much faster exchange rate for the water molecule coordinated to the metal ion, was the most abundant, thus explaining the high relaxivity of the micellar agent. The systemic administration of the micelles into a lipopolysaccharide-induced murine model of acute inflammation successfully demonstrated the ability of the targeting agents to detect the diseased area by T1 contrast enhanced MRI.

Novel Gastrin-Releasing Peptide Receptor Targeted Near-Infrared Fluorescence Dye for Image-Guided Surgery of Prostate Cancer.

PURPOSE: Prostate cancer (PCa), the most widespread male cancer in western countries, is generally eradicated by surgery, especially if localized. However, during surgical procedures, it is not always possible to identify malignant tissues by visual inspection. Among the possible consequences, there is the formation of positive surgical margins, often associated with recurrence. In this work, the gastrin-releasing peptide receptor (GRPR), overexpressed in the prostatic carcinoma and not in healthy tissues or in benign hyperplasia (BPH), is proposed as target molecule to design a novel near-infrared fluorescent (NIRF) probe for image-guided prostatectomy. PROCEDURES: The NIRF dye Sulfo-Cy5.5 was conjugated to a Bombesin-like peptide (BBN), targeting GRPR. The final product, called BBN-Cy5.5, was characterized and tested in vitro on PC-3, DU145, and LnCAP cell lines, using unconjugated Sulfo-Cy5.5 as control. In vivo biodistribution studies were performed by optical imaging in PC-3 tumor-bearing and healthy mice. Finally, simulation of the surgical protocol was carried out. RESULTS: BBN-Cy5.5 showed high water solubility and a good relative quantum yield. The ability of the probe to recognize the GRPR, highly expressed in PC-3 cells, was tested both in vitro and in vivo, where a significant tumor accumulation was achieved 24 h post-injection. Furthermore, a distinguishable fluorescent signal was visible in mice bearing PCa, when the surgery was simulated. By contrast, low signal was found in healthy or BPH-affected mice. CONCLUSIONS: This work proposes a new NIRF probe ideal to target GRPR, biomarker of PCa. The promising data obtained suggest that the dye could allow the real-time intraoperative visualization of prostate cancer.