Detection of Rejection with CMRI in the Clinical Arena
Up to now, most of the clinical applications for detecting cardiac allograft rejection with CMRI are mainly detecting manifestation of the rejection in the tissue or organ levels, viewing allograft rejection as one of the pathological states of the heart [4
]. Reduced ejection fraction (EF) [5
], prolonged T2
], altered T1
, hyperenhancement with delayed enhancement (DE), and elevated Gd-based contrast agent uptake [5
] have been found to be associated with cardiac allograft rejection, probably due to edema, necrosis, and fibrosis resulting from rejection. Decreased myocardial perfusion [5
] and changes in myocardial perfusion reserve have been found in transplant patients suffering from CAV. Combinations of multiparameter CMRI, accessing cine, T1
, perfusion, and DE together, can make a good diagnosis of compromised heart conditions with rejection in one CMRI session [34
]. In addition, 31
P MR chemical shift imaging [37
] has also been found to change in a small cohort of CAV patients.
Although powerful, current CMRI is more useful for confirming rejection diagnosis with noticeable clinical symptoms, or tracking progress of cardiac function improvement upon treatments, because current CMRI is detecting later expression in the rejection cascade, such as edema, necrosis, and fibrosis, when the myocardial injury has already occurred. However, the majority of patients with earlier ACR show no significant changes in EF or standard global systolic functions [5
]. In addition, patients with CAV are usually without angina due to denervation of the graft heart, so they are unaware of CAV development. By the time the clinical symptoms are presented, or depressed global systolic cardiac functions are detected, it is often beyond the point for effective therapy to fully restore the graft health. In addition, many of the CMRI indicators for ill-functioned heart are shared with other pathological conditions of the heart; hence specific diagnosis of rejection with CMRI alone is difficult.
Our goal is to develop more sensitive CMRI methodology that is able to detect earlier or milder phases of allograft rejection, before irreversible tissue damage occurs (ie, before clinical symptoms emerge), to allow opportunities for clinical intervention and therapy. We have employed a two-pronged approach with cellular and functional MRI, to monitor both the immune cell infiltration at the rejecting heart, as well as the earlier local cardiac dysfunction and strain abnormality resulting from rejection, in search for sensitive and reliable noninvasive CMRI parameters for correct diagnosing of rejection in early milder rejection phases before irreversible tissue damage occurs.
Cellular CMRI for Rejection
T-lymphocytes are thought to coordinate both acute and chronic rejection. Macrophages are present in large quantity throughout the entire rejection process. B-lymphocytes, dendritic cells (DCs), as well as endothelial cells all play a role in the rejection process. The presence of these various immune cell types can indicate the status of rejection. Detecting and profiling the temporal and spatial relationships of these cells with CMRI can serve as potential surrogate markers for rejection.
To make the cells of interest detectable with MRI, a substantial differentiation in signal needs to be created between the target cells and the surrounding tissues. This can be achieved by labeling the target cells, such as macrophages and T-lymphocytes, with MR contrast agents. Iron oxide (FenOm) particles have been the most commonly used MR contrast agent for cell tracking, because of their ability to cause large 1H signal perturbation, resulting in considerable hypointensity in T2*-weighted MR images.
Iron oxide nanoparticles are composed of Fe2O3 and Fe3O4 cores, and are usually stabilized by different coating materials, which shield the metallic core and make the particles biocompatible. Coating materials, such as dextran, polyethylene glycol (PEG), or other polysaccharide derivatives, are bio-degradable; some materials, such as styrene-divinyl benzene inert polymers, are not biodegradable. The overall hydrodynamic sizes of the particles and the coating materials not only determine the in vivo lifetime of the particles in the labeled cells and in the blood stream, but also the extent of incorporation of the particles into the cells. Superparamagnetic iron oxide (SPIO) and ultra-small superparamagnetic iron oxide (USPIO) nanoparticles consist of compatible iron core sizes (4–5 nm and 5–8 nm, respectively) and similar surface coating materials, yet different total hydrodynamic sizes (120–180 nm and 15–30 nm). On the other hand, the much larger micrometer-sized particles, MPIO, usually composed of a magnetite core and an inert coat, are around 1 μm to several micrometers. Because of the large core magnetite contents, one single MPIO particle contains more than 1 pg of iron, whereas it takes a few more degrees of magnitudes of SPIO or USPIO particles to achieve the same amount.
The MR labels can be introduced into the target cells via one of two main routes. In the ex vivo or in vitro labeling schemes, the cells of interest isolated from the host or cell lines are tagged with MR markers in culture, and the labeled cells are then introduced into the subject. In the in vivo or in situ labeling schemes, MR labeling agents are administered systemically and the target cells phagocytose MR labels in their natural biological environments without cell isolation or culture.
Cellular Imaging of Macrophages in Acute Rejection
Macrophages, being phagocytotic, can be easily labeled in vivo, or in situ. MR contrast agents present in the circulation can be ingested by circulating monocytes/macrophages, which migrate to the rejection sites [21•
]. One day after in vivo USPIO labeling (), patches of hypointensity can be seen through the left ventricle (LV) and right ventricle (RV) of allograft hearts [22•
]. Dark areas are concentrated foci of iron-laden macrophages, which are confirmed by pathological ED1 staining of macrophages. Macrophage-infiltrated foci appear to be highly heterogeneous in space. Thus, sampling errors are likely to result when probing rejection with biopsy needles. EMB samples are usually taken from anteroseptal wall of the RV, which does not appear to have high concentration of macrophage infiltration. It is not surprising, then, to find EMB sampling that does not reflect the actual cellular infiltration status of the graft. Monitoring rejection with cellular CMRI is not only noninvasive, but also provides a valuable overall three-dimensional whole-volume perspective of rejection.
Fig. 1 T2*-weighted image showing USPIO-labeled immune cell infiltration in transplanted hearts. a, b, Isografts; c, d, Grade III allografts; and e, Grade II allograft. Patches of hypointensity indicate areas with high numbers of USPIO-labeled immune cells. (more ...)
USPIO and SPIO nanoparticles with dextran coating are biocompatible and biodegradable. The externally administered nanoparticles can be digested and incorporated into the systemic iron pool over time. Thus, the hypointensity can be “reversible” depending on the observation time window. Repetitive imaging over a longer time period requires repetitive administration of the nanoparticles prior to imaging. It has been found that the hypointensity reflects rejection status of the grafts. In both cardiac [21•
] and renal transplants [41
], the degree of hypointensity increases as the rejection progresses, decreases upon cyclosporine immunosuppressive treatment, and the extent of hypointensity increases again after withdrawing the immunosuppressive drug [21•
]. The biodegradable SPIO and USPIO are found to be tolerated well, incorporating into body iron pool upon metabolism [43
], and imposing very little safety and toxicity concerns [44
]. A similar type of particles, Feridex, is approved by the US Food and Drug Administration for human use. The clinical translation for cellular imaging with MRI is feasible.
MPIO, the larger micrometer-sized iron-oxide particles, due to its high iron content and special iron core structure that generates large background gradient and local magnetic field perturbation, makes imaging single or few cells in vivo possible [25•
]. Various types of iron-oxide particles can give different but compatible contrast patterns in rejecting hearts [25•
]. With in vivo labeling of MPIO particles, macrophage infiltration foci show punctate and circular dots () at the location comparable to that with USPIO labeling. At earlier rejection phase, macrophage infiltration is more toward epicardium; as the rejection progresses over time, macrophage infiltration penetrates deeper into myocardium tissue. This cellular infiltration progression pattern is confirmed by pathology () and it has not yet been observed by biopsy due to limited sizes of the biopsy tissues. MRI is not only noninvasive, but also can provide an entire whole-volume perspective of the rejection processes over time.
Fig. 2 Contrast patterns with different contrast agent labeling. a–c, In vivo MRI of macrophage accumulation on POD 6 of a an allograft heart with MPIO-particle labeling; b an isograft heart with MPIO-particle labeling; and c an allograft heart with (more ...)
Fig. 3 Temporal progression of ED1+ macrophage infiltration. Optical micrograph (400× magnification) of anti-rat ED1+ immunohistochemical staining sections of allograft hearts obtained on POD 3 a, POD 4 b, POD 5 c, and POD 6 d. The ED1+ cells appear (more ...)
MPIO, with current styrene-divinyl benzene inert polymer coating, are not biodegradable. Although having extremely short blood half-life (minutes), the labels are stable for long periods of time (many months) once ingested into cells, allowing repetitive observation without further administration. After single injection of MPIO particles, only little hypointensity was observed in early days while no or little rejection occurs. As rejection progresses over time, even without further administration of particles, greater areas of more hypointensity are seen while more MPIO-labeled macrophages are recruited into the rejecting graft heart [25•
]. Thus, single MPIO administration permits repetitive longitudinal monitoring of the entire rejection process.
Cellular Imaging of Macrophages in Chronic Rejection
With detailed cellular and molecular mechanisms that still need to be elucidated, CAV remains the major challenge for heart transplantation. MPIO particles, with stable and inert polymer coating and large quantity of iron contents, are suitable for long-term tracking of immune cells in CAV. Single administration of MPIO particles allows longitudinal observation of the same subject over a long period of time for a few months, allowing repetitive imaging of chronic rejection [26•
In our single-gene mismatch PVG transplantation model, CAV develops over a few months of time, with no need for immunosuppressive treatments. It is found that macrophages appear very early in the CAV [26•
], and increase as CAV progresses to be more evident ( and ). Macrophages can be an early marker for CAV [26•
], prior to presentation of clinical symptoms, offering sensitive early detection of CAV.
Fig. 4 Representative MRM images of transplant and native hearts. Punctate regions of hypointensity can be clearly seen in the allograft heart harvested on POD 109 a after in vivo labeling with MPIO. A representative isograft heart harvested on POD 119 is shown (more ...)
Fig. 5 Correlation of MRM and iron staining of MPIO in a POD 94 allograft heart. Image from MRM shows the discrete and circular spots of hypointensity (left panel). These black spots of hypointensity effects are due to the presence of MPIO particles, which was (more ...)
Many iron oxide particles are also designed to contain fluorescent tags (). Multimodality cell tracking probes allow for both MR and optical imaging [9•
]. In addition, MR probes with different colors of fluorescent tags can facilitate molecular and cellular mechanistic investigation for rejection biology.
Fig. 6 Fluorescent microscopic images (×400 magnification) of double immunofluorescence staining on frozen tissue from an allograft on POD 20. a A section taining with anti-rat ED1 mAb (for macrophages; arrows, red). b The same section as Panel A staining (more ...)
Imaging Other Immune Cell Types in Rejection
Non-phagocytotic cells, such as T-lymphocytes, B-lymphocytes, or bone marrow–derived mesenchymal stem cells (MSCs), unlike phagocytotic macrophages, cannot readily take up MRI contrast agents. They cannot be labeled efficiently in vivo or in situ, but need to be labeled ex vivo in culture. The labeling efficiency for T-lymphocytes with currently available iron oxide nanoparticles is low (around 10% to 25%) with simple co-incubation in culture. Some external manipulation is usually necessary to label T cells with sufficient amounts of iron (at least 1 pg per cell), such as electroporation, positively charged transfection agents, attaching to HIV-tat peptide, or receptor-mediated endocytosis [23
We have been developing a new class of iron oxide particles [48
] that can be more readily ingested by the non-phagocytotic cell types, for efficient labeling for cellular MRI. The newly synthesized, PEG-coated, nano-sized iron oxide particle, ITRI-IOP (), can label phagocytic macrophages and DCs, as well as non-phagocytic bone marrow–derived MSCs isolated from rats in vitro with ITRI-IOP without the use of additional agents or manipulation (). The labeling efficiency is as high as 92% to 99%, and the labeled cells show larger T2
* perturbation compared to commercially available USPIO and SPIO particles. In addition, ITRI-IOPs yield comparable hypointensity and imaging quality with MPIO particles when labeling macrophages in vivo. We have been able to label T cells with high efficiency without aiding agents using these particles (unpublished results). ITRI-IOPs and their different classes of derivatives are promising reagents for efficient non-phagocytotic cell labeling for imaging allograft rejection and other cell tracking research.
Fig. 7 Characterization of ITRI-IOP. a Schematic drawing of the PEG-coated ITRI-IOP, and the TEM image of the iron core of the ITRI-IOP particles. b Dynamic light scattering analysis of the hydrodynamic diameter of iron-oxide particles. Peak intensity is provided (more ...)
Fig. 8 Light microscopy of cells labeled with ITRI-IOP. Light microscopy (×200) of a macrophages, b dendritic cells, and c MSCs co-cultured with ITRI-IOP for 24 h, where the brown or light brown color indicates the incorporation of iron-oxide particles (more ...)
One potential limitation of macrophage imaging is that it is difficult to distinguish early ischemic injury and inflammation from rejection [4
]. Using different tags to label T cells and macrophages will allow us to further discern correct cellular profiles throughout the rejection process.
Functional CMRI for Rejection
Global systolic functions and other current CMRI parameters appear to be normal with no significant alternation until the later stage of rejection, when myocardial injury has already taken place. To detect potential early mild local changes in cardiac function without significant changes in global systolic functions, tagging CMRI () is implemented to look for early local changes in wall motion and strains in mild rejection. Tagging CMRI places signal-void grids on tissue by saturating proton spins at designated planes in space prior to the imaging sequence and these grids serve as the material points for tracking heart motion, to mark tissue elongation, stretching, or depression and shortening. Strain analysis can quantify regional ventricular wall motion and tissue deformation in high precision.
Fig. 9 MRI tagging. Tagged images for a native heart (a, f), and the same transplanted hearts shown in (see Wu et al. [22•]). b, g is the same isograft shown in ; c, h is the allograft shown in ; d, i is the allograft shown in (more ...)
Our recent results [22•
] have shown that early changes in ventricular wall motion, like immune cell infiltration, are heterogeneous (). The early changes of regional strains also appear to be highly heterogeneous ( and ). Early mild subtle changes in local strain and ventricular wall motion can be detected while no significant changes in EF and stroke volume are observed. The degrees of local strain changes correlate well with rejection status, even when no changes are found in global systemic functions (). Since cellular infiltration causing local contractile abnormality is not necessarily following the coronary territories, and can happen in any locations of the heart, tagging MRI with high-resolution strain analysis can permit analysis of regional wall motion with high resolution, free from coronary architecture, and is not limited to the conventional 17-segment model (). While overall strain analysis and modeling can be labor intensive, clinically relevant indexes can be extracted () for potential clinical translation.
Fig. 10 Simultaneous tracking of cellular infiltration and function for 3 allografts: a–c, T2*-weighted MRI; d–f, pseudo-coloring of T2*-weighted images with 8-bit signal-intensity scaling; and g–i, Ecc strain maps. Absolute or positive (more ...)
Fig. 11 Mean Ecc a, stroke volume (SV; b), and ejection fraction (EF; c) for native and transplanted hearts: native, isograft, allograft grade I, grade II, grade III, and grade IV. |Ecc|: absolute or positive values of Ecc strain are used without negative signs. (more ...)
Fig. 12 Rejection grading by Ecc probe-point values. a Numbers of “compromised” probe points for each of grade I (G1), grade II (GII), grade III (GIII), and grade IV (GIV) allografts. Probe-points with “compromised” Ecc strain (more ...)
Our findings in regional strain abnormality without alternation in global systolic functions (EF) in early rejection are consistent with recent development of detecting strain and strain rate changes in rejection with echocardiography [49
There is encouraging addition of accuracy in diagnosing rejection when simultaneously assessing both cellular imaging of macrophage infiltration and regional functional strain analysis (). While there are fine individual variations of the degrees of cellular infiltration and of local strain abnormality, individuals with more immune cell infiltration show greater degrees of compromised strains (), reflecting finer degrees of deterioration. Simultaneous coupling of cellular and functional aspects of rejection adds further accuracy and sensitivity for detecting rejection. Our two-pronged approach with cellular and functional MRI for both cellular infiltration and strains provides a promising potential for noninvasive rejection detection. More detailed analysis with larger sample sizes might lead to possible finer staging of rejection within the current mild to moderate rejection grades (I, IIA, and IIB) defined by EMB, and can offer a more precise and sensitive method for assessing ACR.
Fig. 13 Quantitation for USPIO cell infiltration. a Percentage of contrast-containing volume from T2*-weighted images for isografts, grade I, grade II, grade III, and grade IV allografts. b Plot percentage of contrast-containing volume against mean Ecc strain (more ...)