The central role of cell death in cardiovascular disease makes the detailed understanding of this process imperative.11, 12
We present here a novel DNA-binding agent, Gd-TO, to image necrotic cell death in vivo
by MRI. The specificity of Gd-TO for acute necrotic cell death is demonstrated in a mouse model of myocardial infarction. We show that the agent has the unique ability to image both the mechanism and chronicity of cell death through a novel and well-elucidated molecular mechanism. While we focus in the current study on a mouse model of myocardial infarction, the utility of the agent is extremely broad and it can be used to image necrotic cell death in any cardiovascular condition. A versatile, well-characterized and effective platform for the imaging of acute necrotic cell death is thus presented.
The kinetics and mechanisms of cell death in the myocardium, remain incompletely understood.12, 13
Moreover, the response of serological biomarkers such as troponin to injury within a solid organ can be highly delayed and nonlinear.14
In contrast, the use of a vital imaging agent such as Gd-TO provides a direct and spatially resolved readout of cell death at the local level. Gd-TO uptake in this study was present within 2 hours of myocardial infarction and peaked between 9–18 hours of injury. This suggests that cell rupture and significant changes in membrane permeability begin within 1–2 hours of infarction. Full disintegration and rupture of the cell, however, takes many more hours to occur and maximal uptake of Gd-TO thus occurred 9–18 hours after infarction. This time course is consistent with previous histological data using an antimyosin antibody.15
Notwithstanding differences in human and murine pathophysiology, the kinetics of Gd-TO accumulation provide valuable insights into the kinetics of serological biomarkers such as troponin. Troponin elevation in patients with acute coronary syndromes is frequently detected only 6–12 hours after injury,14
well after the initial uptake of Gd-TO in this study. This suggests that the release of troponin into the extracellular space is a late event in cardiomyocyte necrosis, and that elevations in serum troponin will be seen only once profound disintegration of the cardiomyocyte membrane has occurred.
The response of the inflammatory system to the release of cellular DNA has been extensively studied.16, 17
Free DNA in its own right serves as a danger signal, stimulating an inflammatory response.16
DNA also interacts with toll-like receptors on monocytes,18
promoting phagocytosis. In addition, several opsonins that bind DNA and promote its phacocytosis have been identified including C1q,19
ficolin-2 and 3,21
and histidine-rich glycoprotein.23
These and other mechanisms result in a robust monocyte infiltrate within the infarct, which in mice reaches full threshold approximately 24 hours after injury.24, 25
The absence of Gd-TO uptake 72–96 hours after myocardial infarction suggests that the removal of necrotic cell debris by infiltrating monocytes is completed within 2–3 days, consistent with prior histological studies.24
Interestingly, this time point corresponds to the transition of the monocytes infiltrating infarcted myocardium from highly degradative lys6C-high monocytes to more reparative lys6C-low monocytes.25
Antibodies targeted to specific antigens, such as myosin,26
can be used to image membrane integrity. However, antibodies are far too large for renal elimination and frequently show high non-specific hepatic accumulation. The simultaneous use of radiolabeled annexin and anti-myosin antibodies,27, 28
although of value, is thus significantly limited by these factors. In contrast, the molecular weights of TO and the complete Gd-TO molecule are 305 Da and 1110 Da, respectively. Gd-TO's physical properties (small size, water solubility, lack of albumin binding) likely contribute to its rapid clearance from the blood and its low tissue retention at 24 hours (), a prerequisite for the clinical translation of any gadolinium-based agent. Imaging of Gd-TO uptake was performed 2–3 hours after injection in this proof-of-principle study. However, images acquired in our study one hour after injection suggest that this time point would also be highly suitable for the imaging of Gd-TO.
The development of T2
weighted MRI sequences to image tissue edema has been a major advance.29, 30
hyperintensity is a non-specific signature, and cannot differentiate acute (less than 72 hours) from subacute (less than 6 weeks) injury. The use of serological biomarkers such as troponin is frequently helpful, but not definitive, in these situations. Regions of acute and subacute injury, for instance, can frequently co-exist and cannot be easily distinguished from each other using T2
weighted MRI and serological biomarkers. The availability of a single definitive imaging test to differentiate acute from subacute injury would thus be a valuable advance. Moreover, the use of Gd-TO to study the mechanism, kinetics and immune response to cell death has the potential to yield valuable insights into myocardial injury and result in improved therapeutic strategies.
Delayed (late) enhancement imaging of extracellular chelates in the myocardium is based on changes in the extracellular volume of distribution of the chelate, and is a non-specific finding seen in both acute and chronic injury.31, 32
Gadolinium chelates such as gadoporphyrin, which preferentially accumulate in necrotic tissue have been developed.33, 34
However, the mechanism of uptake of these agents is completely non-specific and is likely related to binding to a variety of proteins and connective tissue elements. In contrast, the uptake of Gd-TO occurs via a specific and well-elucidated molecular mechanism and is thus able to provide novel insights into the kinetics of cell death and clearance in infarcted myocardium.
Molecular MRI of apoptosis in vivo
has been performed with several constructs, most notably the superparamagnetic nanoparticle AnxCLIO-Cy5.5. In an initial proof-of-principle study in the heart, this agent was used alone and was thus not able to distinguish apoptosis from necrosis.35
In a subsequent study, delayed enhancement of gadolinium (Gd-DTPA) was used in conjunction with AnxCLIO-Cy5.5 to attempt to resolve the various forms of cell death in the myocardium.36
While of value, the non-specific nature and limitations of delayed enhancement imaging have been discussed above. The availability of AnxCLIO-Cy5.5 and Gd-TO now makes it possible to replicate, in the in vivo
setting, the robust dual fluorochrome approach used during intravital microscopy and flow cytometry.
In conclusion, the work presented here is, to the best of our knowledge, the first demonstration of molecular MRI with a multimodal vital imaging agent. We show that Gd-TO is able to image the mechanism of cell death as well as the evolution and clearance of necrotic cells in acute ischemia. In addition, we show that inflammatory cells do not take up Gd-TO, making the agent suitable for imaging necrotic cell death in highly inflammatory milieus such as infarcted myocardium, myocarditis and transplant rejection. Gd-TO does not require the synthesis of new biological materials and is well suited to synthesis at the scale needed for large animal and human studies. The structure, binding and mechanism of uptake of Gd-TO are all well understood, and it is well eliminated. The potential of Gd-TO, or a radioactive TO analogue, to undergo successful clinical translation is thus high.