Monocyte MRI Relaxation Rates Are Regulated by Extracellular Iron and Hepcidin.

Many chronic inflammatory conditions are mediated by an increase in the number of monocytes in peripheral circulation, differentiation of monocytes to macrophages, and different macrophage subpopulations during pro- and anti-inflammatory stages of tissue injury. When hepcidin secretion is stimulated during inflammation, the iron export protein ferroportin is targeted for degradation on a limited number of cell types, including monocytes and macrophages. Such changes in monocyte iron metabolism raise the possibility of non-invasively tracking the activity of these immune cells using magnetic resonance imaging (MRI). We hypothesized that hepcidin-mediated changes in monocyte iron regulation influence both cellular iron content and MRI relaxation rates. In response to varying conditions of extracellular iron supplementation, ferroportin protein levels in human THP-1 monocytes decreased two- to eightfold, consistent with paracrine/autocrine regulation of iron export. Following hepcidin treatment, ferroportin protein levels further decreased two- to fourfold. This was accompanied by an approximately twofold increase in total transverse relaxation rate, R2*, compared to non-supplemented cells. A positive correlation between total cellular iron content and R2* improved from moderate to strong in the presence of hepcidin. These findings suggest that hepcidin-mediated changes detected in monocytes using MRI could be valuable for in vivo cell tracking of inflammatory responses.

Investigating the Relationship between Transverse Relaxation Rate (R2) and Interecho Time in MagA-Expressing, Iron-Labeled Cells.

Reporter gene-based labeling of cells with iron is an emerging method of providing magnetic resonance imaging contrast for long-term cell tracking and monitoring cellular activities. This report investigates 9.4 T nuclear magnetic resonance properties of mammalian cells overexpressing MagA, a putative iron transport protein from magnetotactic bacteria. MagA-expressing MDA-MB-435 cells were cultured in the presence and absence of iron supplementation and compared to the untransfected control. The relationship between the transverse relaxation rate (R2) and interecho time was investigated using the Carr-Purcell-Meiboom-Gill sequence. This relationship was analyzed using a model based on water diffusion in weak magnetic field inhomogeneities (Jensen-Chandra model) as well as a fast-exchange model (Luz-Meiboom model). Increases in R2 with increasing interecho time were larger in the iron-supplemented, MagA-expressing cells compared to other cells. The dependence of R2 on interecho time in these iron-supplemented, MagA-expressing cells was better represented by the Jensen-Chandra model compared to the Luz-Meiboom model, whereas the Luz-Meiboom model performed better for the remaining cell types. Our findings provide an estimate of the distance scale of microscopic magnetic field variations in MagA-expressing cells, which is thought to be related to the size of iron-containing vesicles.

Hepcidin-mediated Iron Regulation in P19 Cells is Detectable by Magnetic Resonance Imaging.

Magnetic resonance imaging can be used to track cellular activities in the body using iron-based contrast agents. However, multiple intrinsic cellular iron handling mechanisms may also influence the detection of magnetic resonance (MR) contrast: a need to differentiate among those mechanisms exists. In hepcidin-mediated inflammation, for example, downregulation of iron export in monocytes and macrophages involves post-translational degradation of ferroportin. We examined the influence of hepcidin endocrine activity on iron regulation and MR transverse relaxation rates in multi-potent P19 cells, which display high iron import and export activities, similar to alternatively-activated macrophages. Iron import and export were examined in cultured P19 cells in the presence and absence of iron-supplemented medium, respectively. Western blots indicated the levels of transferrin receptor, ferroportin and ubiquitin in the presence and absence of extracellular hepcidin. Total cellular iron was measured by inductively-coupled plasma mass spectrometry and correlated to transverse relaxation rates at 3 Tesla using a gelatin phantom. Under varying conditions of iron supplementation, the level of ferroportin in P19 cells responds to hepcidin regulation, consistent with degradation through a ubiquitin-mediated pathway. This response of P19 cells to hepcidin is similar to that of classically-activated macrophages. The correlation between total cellular iron content and MR transverse relaxation rates was different in hepcidin-treated and untreated P19 cells: slope, Pearson correlation coefficient and relaxation rate were all affected. These findings may provide a tool to non-invasively distinguish changes in endogenous iron contrast arising from hepcidin-ferroportin interactions, with potential utility in monitoring of different macrophage phenotypes involved in pro- and anti-inflammatory signaling. In addition, this work demonstrates that transverse relaxivity is not only influenced by the amount of cellular iron but also by its metabolism.

MagA expression attenuates iron export activity in undifferentiated multipotent P19 cells.

Magnetic resonance imaging (MRI) is a non-invasive imaging modality used in longitudinal cell tracking. Previous studies suggest that MagA, a putative iron transport protein from magnetotactic bacteria, is a useful gene-based magnetic resonance contrast agent. Hemagglutinin-tagged MagA was stably expressed in undifferentiated embryonic mouse teratocarcinoma, multipotent P19 cells to provide a suitable model for tracking these cells during differentiation. Western blot and immunocytochemistry confirmed the expression and membrane localization of MagA in P19 cells. Surprisingly, elemental iron analysis using inductively-coupled plasma mass spectrometry revealed significant iron uptake in both parental and MagA-expressing P19 cells, cultured in the presence of iron-supplemented medium. Withdrawal of this extracellular iron supplement revealed unexpected iron export activity in P19 cells, which MagA expression attenuated. The influence of iron supplementation on parental and MagA-expressing cells was not reflected by longitudinal relaxation rates. Measurement of transverse relaxation rates (R2* and R2) reflected changes in total cellular iron content but did not clearly distinguish MagA-expressing cells from the parental cell type, despite significant differences in the uptake and retention of total cellular iron. Unlike other cell types, the reversible component R2' (R2* ‒ R2) provided only a moderately strong correlation to amount of cellular iron, normalized to amount of protein. This is the first report to characterize MagA expression in a previously unrecognized iron exporting cell type. The interplay between contrast gene expression and systemic iron metabolism substantiates the potential for diverting cellular iron toward the formation of a novel iron compartment, however rudimentary when using a single magnetotactic bacterial gene expression system like magA. Since relatively few mammalian cells export iron, the P19 cell line provides a tractable model of ferroportin activity, suitable for magnetic resonance analysis of key iron-handling activities and their influence on gene-based MRI contrast.

Expression analysis of a novel p75(NTR) signaling protein, which regulates cell cycle progression and apoptosis.

Neurotrophin receptor-interacting MAGE (NRAGE) is the most recently identified p75 neurotrophin receptor (p75(NTR)) intracellular binding protein. Previously, NRAGE over-expression was shown to mediate cell cycle arrest and facilitate nerve growth factor (NGF) dependent apoptosis of sympathetic neuroblasts in a p75(NTR) specific manner. Here we have examined the temporal and spatial expression patterns of NRAGE over the course of murine embryogenesis to determine whether NRAGE's expression is consistent with its proposed functions. We demonstrate that NRAGE mRNA and protein are expressed throughout embryonic and adult tissues. The mRNA is constitutively expressed within each tissue across development. However, expression of NRAGE protein displays a tight temporal tissue specific regulation. During early CNS development, NRAGE protein is expressed throughout the neural tube, but by later stages of neurogenesis, NRAGE protein is restricted within the ventricular zone, subplate and cortical plate. Moreover, NRAGE protein expression is limited to proliferative neural subpopulations as we fail to detect NRAGE expression co-localized with mature/differentiation associated neuronal markers. Interestingly, NRAGE's expression is not restricted solely to areas of p75(NTR) expression suggesting that NRAGE may mediate proliferation and/or apoptosis from other environmental signals in addition to NGF within the CNS. Our data support previously characterized roles for NRAGE as a mediator of precursor apoptosis and a repressor of cell cycle progression in neural development.

The Interface Between Iron Metabolism and Gene-Based Iron Contrast for MRI.

Using a gene-based approach to track cellular and molecular activity with magnetic resonance imaging (MRI) has many advantages. The strong correlation between transverse relaxation rates and total cellular iron content provides a basis for developing sensitive and quantitative detection of MRI reporter gene expression. In addition to biophysical concepts, general features of mammalian iron regulation add valuable context for interpreting molecular MRI predicated on gene-based iron labeling. With particular reference to the potential of magnetotactic bacterial gene expression as a magnetic resonance (MR) contrast agent for mammalian cell tracking, studies in different cell culture models highlight the influence of intrinsic iron regulation on the MRI signal. The interplay between dynamic regulation of mammalian iron metabolism and expression systems designed to sequester iron biominerals for MRI is presented from the perspective of their potential influence on MR image interpretation.

Biophysical features of MagA expression in mammalian cells: implications for MRI contrast.

We compared overexpression of the magnetotactic bacterial gene MagA with the modified mammalian ferritin genes HF + LF, in which both heavy and light subunits lack iron response elements. Whereas both expression systems have been proposed for use in non-invasive, magnetic resonance (MR) reporter gene expression, limited information is available regarding their relative potential for providing gene-based contrast. Measurements of MR relaxation rates in these expression systems are important for optimizing cell detection and specificity, for developing quantification methods, and for refinement of gene-based iron contrast using magnetosome associated genes. We measured the total transverse relaxation rate (R2*), its irreversible and reversible components (R2 and R2', respectively) and the longitudinal relaxation rate (R1) in MDA-MB-435 tumor cells. Clonal lines overexpressing MagA and HF + LF were cultured in the presence and absence of iron supplementation, and mounted in a spherical phantom for relaxation mapping at 3 Tesla. In addition to MR measures, cellular changes in iron and zinc were evaluated by inductively coupled plasma mass spectrometry, in ATP by luciferase bioluminescence and in transferrin receptor by Western blot. Only transverse relaxation rates were significantly higher in iron-supplemented, MagA- and HF + LF-expressing cells compared to non-supplemented cells and the parental control. R2* provided the greatest absolute difference and R2' showed the greatest relative difference, consistent with the notion that R2' may be a more specific indicator of iron-based contrast than R2, as observed in brain tissue. Iron supplementation of MagA- and HF + LF-expressing cells increased the iron/zinc ratio approximately 20-fold, while transferrin receptor expression decreased approximately 10-fold. Level of ATP was similar across all cell types and culture conditions. These results highlight the potential of magnetotactic bacterial gene expression for improving MR contrast.

Imaging tumor growth non-invasively using expression of MagA or modified ferritin subunits to augment intracellular contrast for repetitive MRI.

PURPOSE: The bacterial gene MagA imparts magnetic properties to mammalian cells and provides a basis for cell tracking by magnetic resonance imaging (MRI). In a mouse model of tumor growth from transplanted cells, we used repetitive MRI to demonstrate the in vivo imaging potential of MagA expression relative to a modified ferritin overexpression system, lacking regulation through iron response elements (HF + LF). PROCEDURES: Subcutaneous tumor xenografts were monitored weekly from days 2 to 34 post-injection. Small animal MRI employed balanced steady-state free precession. Imaging was correlated with tumor histology using hematoxylin, Prussian Blue, Ki-67, and BS-1 lectin. RESULTS: Tumor heterogeneity with respect to tissue morphology and magnetic resonance (MR) contrast was apparent within a week of cell transplantation. In MagA- and HF + LF-expressing tumors, MR contrast enhancement was recorded up to day 20 post-injection and 0.073-cm(3) tumor volumes. MagA-expressing tumors showed increases in both quantity and quality of MR contrast as measured by fractional void volume and contrast-to-noise ratio, respectively. MR contrast in both MagA- and HF + LF-expressing tumors was maximal by day 13, doubling fractional void volume 1 week ahead of controls. CONCLUSIONS: MagA- and HF + LF-expressing tumor xenografts augment MR contrast after 1 week of growth. MagA expression increases MR contrast within days of cell transplantation and provides MR contrast comparable to HF + LF. MagA has utility for monitoring cell growth and differentiation, with potential for in vivo detection of reporter gene expression using MRI.

Using the magnetosome to model effective gene-based contrast for magnetic resonance imaging.

Formation of iron biominerals is a naturally occurring phenomenon, particularly among magnetotactic bacteria which produce magnetite (Fe(3) O(4) ) in a subcellular compartment termed the magnetosome. Under the control of numerous genes, the magnetosome serves as a model upon which to (1) develop gene-based contrast in mammalian cells and (2) provide a mechanism for reporter gene expression in magnetic resonance imaging (MRI). There are two main components to the magnetosome: the biomineral and the lipid bilayer that surrounds it. Both are essential for magnetotaxis in a variety of magnetotactic bacteria, but nonessential for cell survival. Through comparative genome analysis, a subset of genes characteristic of the magnetotactic phenotype has been found both within and outside a magnetosome genomic island. The functions of magnetosome-associated proteins reflect the complex nature of this intracellular structure and include vesicle formation, cytoskeletal attachment, iron transport, and crystallization. Examination of magnetosome genes and structure indicates a protein-directed and stepwise assembly of the magnetosome compartment. Attachment of magnetosomes along a cytoskeletal filament aligns the magnetic particles such that the cell may be propelled along an external magnetic field. Interest in this form of magnetotaxis has prompted research in several areas of medicine, including magnetotactic bacterial targeting of tumors, MR-guided movement of magnetosome-bearing cells through vessels and molecular imaging of mammalian cells using MRI, and its hybrid modalities. The potential adaptation of magnetosome genes for noninvasive medical imaging provides new opportunities for development of reporter gene expression for MRI.

Magnetic resonance imaging of cells overexpressing MagA, an endogenous contrast agent for live cell imaging.

Molecular imaging with magnetic resonance imaging (MRI) may benefit from the ferrimagnetic properties of magnetosomes, membrane-enclosed iron biominerals whose formation in magnetotactic bacteria is encoded by multiple genes. One such gene is MagA, a putative iron transporter. We have examined expression of MagA in mouse neuroblastoma N2A cells and characterized their response to iron loading and cellular imaging by MRI. MagA expression augmented both Prussian blue staining and the elemental iron content of N2A cells, without altering cell proliferation, in cultures grown in the presence of iron supplements. Despite evidence for iron incorporation in both MagA and a variant, MagAE137V, only MagA expression produced intracellular contrast detectable by MRI at 11 Tesla. We used this stable expression system to model a new sequence for cellular imaging with MRI, using the difference between gradient and spin echo images to distinguish cells from artifacts in the field of view. Our results show that MagA activity in mammalian cells responds to iron supplementation and functions as a contrast agent that can be deactivated by a single point mutation. We conclude that MagA is a candidate MRI reporter gene that can exploit more fully the superior resolution of MRI in noninvasive medical imaging.