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Molecular imaging represents a targeted approach to non-invasively assess biologic (both physiologic and pathologic) processes in vivo. Ideally the goal of molecular imaging is not just to provide diagnostic and prognostic information based on identification of the molecular events associated with a pathological process, but rather to guide individually tailored pharmacological, cell-based or genetic therapeutic regimens. This article reviews the recent advances in myocardial molecular imaging in the context of the cardiovascular processes of angiogenesis, apoptosis, inflammation and ventricular remodeling. The focus is on radiotracer-based SPECT and PET molecular imaging approaches.
As a greater understanding of the complex molecular interactions that take place within an organism both in the physiologic as well as pathologic states develops, there is an opportunity to design more precise and effective medical therapies that carry less of a burden of side effects and invasive injuries. This evolution of truly individualized health care will require enough knowledge of an individual's genome and proteome to model molecular interactions from levels of gene expression to the complex milieu and kinetics of protein expression and post-translational modification that occur in physiologic and pathologic processes. New therapies that target specific molecular pathways can then be developed to affect outcomes before disease burden affects the health and productivity of an individual in question. To track both disease and intervention, a parallel engineering of tools to image specific molecular events must take place that allows assessment of the in vivo state before and after any intervention is carried out. This article will review the molecular-based nuclear imaging approaches for evaluation of critical processes within the myocardium, such as; angiogenesis, apoptosis, inflammation, and ventricular remodeling.
The application of imaging using biologically targeted markers (i.e. molecular imaging) has a number of requirements1. Selection of a molecular target that adequately represents the process being studied is critical to the specificity of any imaging approach. The next requirement is a readily synthesizable probe that binds to the target molecule with a high degree of specificity. Lastly, an imaging technology that provides the best combination of sensitivity and resolution (both spatial and temporal) to identify and localize the probe within the target organ system must be available. Molecular imaging approaches are currently being developed for most of the imaging modalities; including nuclear, magnetic resonance, X-ray computed tomography (CT), optical fluorescence, bioluminescence, and ultrasound2. Though each modality carries strengths and weaknesses, the practical limitations of cost and wide-spread availability will likely be what allow any modality to be adapted for clinical use. The focus of this paper will be in the specific applications of the nuclear imaging approaches.
Single photon emission computed tomography (SPECT) and positron emission tomography (PET) are imaging techniques that make use of radiolabeled probes and have been used for over three decades. Radiolabeling has the unique advantage of augmenting low signal intensity objects. For example, PET can detect picomolar and nanomolar concentrations of a molecule of interest2. Though SPECT offers the advantage of decreased cost and wide spread availability, PET offers the advantages of increased sensitivity with the ability to quantitate as well as repetitively image through tracers with ultra short half-lives. Traditionally, nuclear imaging modalities have been limited by attenuation artifacts from soft tissue and partial volume effects. More recent systems combining CT imaging with either SPECT or PET have allowed for attenuation correction, leading to improved imaging quantification.
The article will review several important areas of active cardiovascular research in which nuclear-based molecular imaging have been used; including the processes of angiogenesis, apoptosis, inflammation, and ventricular remodeling. Key molecular events or signaling proteins involved in each process that were identified through basic research have served as targets for imaging. Some molecular signals overlap between biological processes which emphasizes the importance of understanding the setting in which a molecular event takes place.
Atherosclerotic disease can lead to chronic ischemia in areas of myocardium, which may stimulate an angiogenic response in order to restore perfusion. Angiogenesis is defined as the process of sprouting new capillaries from preexisting microvessels3. There is a great interest in understanding the processes of angiogenesis in order to design therapeutic treatments that allow revascularization through a stimulated angiogenic response. This angiogenic process often occurs in association with arteriogenesis, which represents the remodeling of larger preexisting vascular channels or collateral vessels feeding the microvascular network. The goal for any revascularization strategy would be to initiate angiogenesis and arteriogenesis in order to create an effective blood delivery systems. There is a large body of literature devoted to understanding these phenomena4. Angiogenesis appears to be stimulated by external processes such as ischemia, hypoxia, inflammation, and shear stress. Several cell types are involved in the process, including endothelial cells, smooth muscle cells, blood derived macrophages, circulating stem cells, and the interaction of these cells within the tissue of extracellular matrix proteins. Potential targets for molecular imaging of angiogenesis can be divided into three major categories5. Non-endothelial targets like molecules associated with monocytes, macrophages, and stem cells, fall into one category. The second category includes endothelial cell targets like vascular endothelial growth factor (VEGF), integrins, CD13, and syndecan-4. Extracellular matrix proteins compose the final group of molecules that might serve as targets.
Hypoxia, the imbalance between oxygen delivery and demand in a given tissue, can be a potent stimulator of angiogenesis. Hypoxic conditions such as myocardial ischemia or infarction result in upregulation of the transcriptional activator hypoxia-inducible factor 1 (HIF-1)6. Upregulation of HIF-1 leads to transcription of vascular endothelial growth factor (VEGF) and the VEGF receptors, Flt-1 (VEGFR-1) and FLK-1 (VEGFR-2) 7-10. When VEGF binds to these receptors on the surface of endothelial cells, a signal is transduced through their tyrosine kinase activity. This initiates a series of processes that results in endothelial cell proliferation, migration, survival, and angiogenesis11. The process also involves the activation molecules like integrins and matrix metalloproteinases as well as the recruitment of inflammatory cells like macrophages.
Integrins are a family of heterodimeric (αβ) cell-surface receptors that mediate divalent cation-dependent cell-cell and cell-matrix adhesion and signaling through tightly regulated interactions with their respective ligands12. During angiogenesis, endothelial cells make use of integrins to adhere to one another and the extracellular matrix to construct and extend new vessels. Integrins are capable of mediating an array of cellular processes, including cell adhesion, migration, proliferation, differentiation and survival via a number of signal transduction pathways13;14. Activation of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) may lead to endothelial cell-induced remodeling of the ECM in response to mechanical stimuli. Specifically, the endothelial cell integrin, αvβ3, allows cells to interact with the ECM in a way that aids in endothelial cell migration15. Through outside-in signaling, integrin αvβ3 also plays a critical roll in the survival of cells undergoing angiogenesis16.
Other molecules have been identified in this schema of endothelial cell activation. Syndecan-4 is a transmembrane heparin sulfate carrying core protein that promotes VEGF to VEGFR binding and signaling via activation of protein kinase C17. CD13 is a cell surface antigen that is expressed in endothelial cells is an aminopeptidase that serves as a membrane-bound metalloproteinase which appears to be essential for capillary tube formation18.
VEGF receptors have been targeted for imaging in models of ischemia-induced angiogenesis. Radiolabeled-VEGF121 has been used to affectively identify angiogenesis in a rabbit model of hindlimb ischemia19. In this study, KDR and Flt-1 receptor expression was increased in the immunohistochemistry analysis of the skeletal muscle, supporting the theoretical hypoxic-driven angiogenic response. One concern of the study is the biodistribution of the radiotracer which revealed 20 fold higher levels in critical organs (liver, kidneys) compared with ischemic limb, which is presumably related to relative VEGFR density in these organ systems.
A PET tracer, 64Cu-6DOTA-VEGF121, was recently developed for imaging angiogenesis in a rat model of myocardial infarction (Figure 1)20. Rats underwent ligation of the left coronary artery and subsequent PET imaging at various time points post myocardial infarction. The investigators hypothesized that this tracer would detect early angiogenic signals because ischemia drives VEGFR expression. Co-registration of images was carried out using CT, and the zone of infarct was demonstrated using 18F-FDG uptake. The study demonstrated that 64Cu-6DOTA-VEGF121- specific signal was present in the infarct region and peaked on day 3 consistent with the changing levels of VEGFR expression in the tissue as analyzed by immunoflourescence microscopy.
Another type of cardiac-specific reporter has been developed as a gene expression system for use in rats with microPET imaging 21. Briefly, the system involves adenovirus delivery of mutated thymidine kinase under the control of a cytomegalovirus promoter that drives expression in myocardial cells. The reporter probe is 18F-FHBG which crosses the myocardial membrane and gets phosphorylated by the thymidine kinase. Phosphorylation essentially traps the 18F-FHGB in the myocardium for subsequent microPET imaging. Early studies revealed that the localized site of the mutated thymidine kinase, HSV1-sr39tk, corresponded closely with that defined by postmortem autoradiography, histology, and immunohistochemistry22. Other studies have demonstrated the feasibility of utilizing a similar reporter system in pigs using a clinical PET scanner23. The reporter system was then linked to VEGF to assess feasibility of developing an approach that links therapy and imaging24. Early experiments with rat embryonic cardiomyocytes revealed a strong correlation that both the mutated thymidine kinase and VEGF were expressed in the same cells. Further studies involved injection of the VEGF/thymidine kinase reporter system in models of ischemia. Using microPET, cardiac transgene expression was assessed and the in vivo imaging correlated well with ex vivo tissue studies for gamma counting, thymidine kinase activity, and VEGF levels. There appeared to be increased capillaries and small blood vessels in the VEGF treated myocardium, however, there was no improvement in perfusion assessed by nitrogen-13 ammonia imaging or metabolism assessed with 18F-FDG imaging. These studies suggest that a reporter system can be developed to help visualize the effectiveness of delivering VEGF gene therapies for stimulation of angiogenesis.
Imaging angiogenic vessels through targeting of αvβ3 integrin was first proposed through a series of magnetic resonance imaging studies using a monoclonal antibody to αvβ3 tagged with a paramagnetic contrast agent25. Such studies were complicated by poor clearance of the tracer from the blood pool. Later studies made use of a number of αvβ3 antagonists that were radiolabeled26;27. These studies made use of the arginine-glycine-aspartate (RGD) - binding sequence on integrins by synthesizing RGD analogues.
An 111In-labeled quinolone (111In-RP748) revealed high affinity and selectivity for αvβ3 integrin in adhesion assays28. Subsequent studies using a cy3-labeled homologue of 111In-RP748, demonstrated preferential binding to activated αvβ3 integrins on endothelial cells in culture , with localization to cell-cell contact points29. Initial studies with this agent focused on imaging tumor angiogenesis, although the first imaging of ischemia-induced myocardial angiogenesis using 111In-RP748 was carried out in rat and canine models of infarction30. In these studies, 111In-RP748 demonstrated favorable kinetics for in vivo SPECT imaging ischemia-induced angiogenesis of the heart. Relative 111In-RP748 activity was markedly increased in the infarcted region acutely and persisted for at least 3 weeks post reperfusion30;31. Therefore, targeted imaging with 111In-RP748 has demonstrated integrin αvβ3 activation early post infarction, suggesting a role for this technique in early detection of angiogenesis as well as for detection of chronic ischemia (Figure 2)31.
Other experiments, utilizing a 99mTc-labeled peptide, NC100692, in the rodent model of hindlimb ischemia for targeting of αvβ3 integrin have also been carried out and support the value of integrin imaging in models of peripheral arterial disease32. The recent imaging of αvβ3 integrin by the PET imaging tracer, 18F-Galakto-RGD, in a patient with a transmural myocardial infarction two weeks prior demonstrates the feasibility of detecting angiogenesis in the myocardium in humans33.
Apoptosis is the physiological process of programmed cell death, whereby organisms selectively target cells to be eliminated when they are no longer needed. The cardiovascular pathologies of cardiomyopathy, heart failure, myocarditis, and myocardial infarction are associated with increased levels of apoptosis, particularly in the myocyte. On the other hand, a subset of cell death that occurs as an outcome of these pathological processes outside of programmed cellular mechanisms is termed necrosis. A recent study evaluating a role of apoptosis and necrosis in the setting of acute myocardial infarction revealed a potential therapeutic role for cyclosporine34. The intervention is hypothesized to minimize peri-infarct, reperfusion-related cell death that takes place in the setting of revascularization. It is estimated that 30% of cardiomyocytes in the injured myocardium become apoptotic as a result of ischemia reperfusion injury, and animal models of acute infarction demonstrate that up to 50% of the final size of the infarct can be related to lethal reperfusion injury35;36. In other animal studies, inhibition of apoptosis with caspase-inhibitors is cardioprotective37-39. There is also some data to suggest early apoptosis may be the pathological substrate that leads from ischemia to necrosis40. An ability to assess cell-death anywhere along the spectrum of apoptosis to necrosis would allow investigators to fine tune a therapeutic regimen and optimize the outcome. In targeting these pathologies for new interventions, it has become apparent that better in vivo imaging techniques for detection of apoptosis will be required.
The earliest studies of apoptosis evolved around histological assessment of the cells undergoing cell death. The earliest descriptions included the microscopic visualization of chromatin condensation, dissolution of nuclear membrane, nuclear shrinkage, and formation of apoptotic bodies that were cleared by phagocytic cells41-43. Over time it became clear that programmed cell death is central to the development and maintenance of homeostasis of a wide array of organisms44. In addition, cell death plays a role in the pathology of various disease states45. Depending on the initiating signals, there are two major pathways for cell death; intrinsic and extrinsic46. The intrinsic pathway is generated from within the cell through DNA damage, mitochondrial signals, and oncogene activation, leading to activation of caspase enzymes, cysteine proteases that cleave after aspartate residues. The extrinsic pathway is initiated through extracellular signals that target cell membrane receptors like Fas, a death receptor. The culmination of this event through either pathway is the activation of a key effector, caspase-347.
Soon after the activation of caspase-3, the energy-dependent asymmetric distribution of phospholipids that enables the definition of various subregions within the lipid bilayer of cell membranes is lost. This leads to increased phosphatidyl serine (PS) on the outer cell membrane from its typical location on the inner cell membrane48. In part, this is the result of increased calcium levels and decreased amounts of ATP that block the translocase enzyme responsible for maintenance of PS. The exposure of PS on the surface of the cell makes it a target for binding the protein, Annexin V49. Annexin V binds to PS in a calcium dependent manner. This has lead to an in vitro assay of fas-ligand initiated cell death through binding of annexin V48;50.
The first application of annexin binding to identify phosphatidyl serine on the surface of cells in a cardiovascular model came from a mouse model of acute myocardial infarction51. In this study, the left anterior descending coronary artery of a series of mice was ligated shortly after the injection of biotinylated annexin V. Immunohistochemical analysis of the tissue distal to the site of ligation revealed annexin A5 binding in an area of cell death. DNA laddering confirmed programmed cell death to be occurring in the same region as the annexin A5 binding.
As myocardial ischemia or infarction persists, cells move from early apoptotic signals to complete necrosis. Breakdown of mitochondrial respiration and loss membrane potential lead to the accumulation of calcium in the mitochondria of infarcted or severely injured myocardium52;53. With loss of membrane potential cellular structures also begin to dissipate. Positively charged histones and other organelle proteins are exposed from the protection of their membrane barriers. These changes in early necrotic tissue have been utilized for imaging techniques that seek to identify early necrosis in acute myocardial infarctions and are discussed in more detail below.
Initial studies utilizing annexin A5 for imaging purposes involved 99mTc labeling. The goal was to image the distribution of cells expressing PS noninvasively with a standard gamma camera. Radiolabeling involved derivatization of annexin A5 with hydrazinonicotinamine (HYNIC), which binds to reduced 99mTc54. The initial studies were carried out in mice with fulminant hepatic apoptosis through the injection of an anti-Fas antibody, which initiates an apoptotic cascade, particularly in hepatocytes55. Concomitant TUNEL studies confirmed localization of annexin A5 with apoptotic cells.
99mTc-labeled annexin V was utilized in humans to detect in vivo cell death in patients presenting with myocardial infarction (Figure 3)56. Patients presenting with their first myocardial infarction within 6 hours of symptom onset underwent standard revascularization with percutaneous intervention. Within 2 hours of revascularization, SPECT imaging was performed utilizing 99mTc-labeled annexin V. This was followed by perfusion imaging 6-8 weeks after discharge using 99mTc-sestamibi. Regional retention of 99mTc-labeled annexin V correlated with the perfusion defect identified 6-8 weeks after discharge, providing a proof of concept that Annexin-V imaging can be utilized for noninvasive detection of myocardial cell death.
Annexin V imaging has also been used to differentiate between benign and malignant cardiac tumors given the high proliferation and cell death rates associated with malignancy57.
Heart transplant rejection is characterized by perivascular and interstitial mononuclear inflammatory infiltrates associated with myocyte apoptosis and necrosis58. In a study of 18 patients undergoing apoptotic imaging within 1 year of cardiac transplantation, Annexin-V retention correlated with the severity of rejection59. Patients with a negative scan had a concomitant negative biopsy. Of the five patients with a positive scan, three patients demonstrated regional uptake while two patients demonstrated diffuse uptake. The Annexin-V scans correlated to the degree of severity of rejection by biopsy specimens. The authors suggested that serial annexin V imaging for apoptotic cells could be used as a surrogate for detection of allograft rejection in place of serial biopsies in patients following heart transplantation.
Myocarditis is another area of pathological condition where apoptosis is known to occur60. In a rat model of autoimmune myocarditis, 99mTc labeled annexin V retention corresponded to histological TUNEL staining for areas of myocardial apoptosis. Interestingly, these areas could be differentiated from areas of inflammation by 14C-labeled deoxyglucose that corresponded to foci of inflammation. This suggests that one could differentiate between inflammation and active apoptosis with molecular imaging. To date, no studies have attempted to employ this technique in conjunction with FDG-PET in human cases of myocarditis.
Because phosphatidyl serine can be exposed on the surface of cells in physiologic conditions other than apoptosis, there is interest in developing more specific apoptosis tracers. Recently, caspase-3 inhibitors have been synthesized and labeled with 18F as potential PET tracers for in vivo imaging of apoptosis (Figure 3)61;62. These caspase-3 targeted tracers have shown favorable biodistribution and clearance. MicroPET imaging in a murine model of hepatic apoptosis has shown specificity of the tracer to the liver, however, more studies are needed to assess binding relative to activated caspase density. In addition, further analysis in cardiovascular models will be necessary to determine feasibility of utilizing this new class of tracers for cardiac applications in human.
The in vivo noninvasive detection myocardial infarction will allow for early diagnosis and treatment in patients when electrocardiographic changes may not be evident or when biomarkers may not distinguish between ischemic injury associated with acute plaque rupture that may be treatable with mechanical revascularization versus unstable angina and demand related ischemia. In addition to visualizing apoptosis, several studies have demonstrated that certain agents allow for the visualization of ongoing myocardial necrosis as a mechanism of identifying acute infarction potentially even in the presence of prior myocardial infarction. 99mTc-labeled pyrophosphate has been shown to bind to areas of necrosis and is thought to bind exposed mitochondrial calcium52;53. 99mTc-pyrophosphate has a moderate degree of sensitivity for acute infarction depending on the presence of Q wave infarction or a non ST elevation infarction63. The specificity for acute myocardial infarction is considered to be between 60% to 80%. The primary reason why 99mTc-pyrophosphate has not gained widespread clinical use is the limitation in the detection of early infarction. In fact, depending of the residual degree of perfusion to the infarct zone, the test may not be positive for the first 24 hours.
99mTc-glucarate imaging provides an alternative to 99mTc-pyrophosphate imaging for the detection of acute infarction53. 99mTc-glucarate enters the necrotic cells by passive diffusion following breakdown of the sarcolemma, and binds to exposed histones in the nucleus of the myocytes. Canine models for ischemia and infarction reveal a high affinity of 99m Tc-glucarate for necrotic tissue over ischemic but viable myocardium64. In a rabbit model of infarction, 99mTc-glucarate did not accumulate in areas of ischemia and could be imaged in areas of infarction as early as 10 minutes post reperfusion and within 30-60 minutes in non-reperfused zones65. Initial data in patients revealed that 99mTc-glucarate is able to noninvasively diagnose myocardial infarction in patients presenting with chest pain with a sensitivity that is dependent on the onset of symptoms, specifically within the first 9 hours of symptom onset 66. 99mTc-glucarate does have a rapid blood clearance and good target to background signal. 99mTc-glucarate imaging is currently under investigation as a tool to detect early infarction in a number of clinical trials.
Inflammation plays an important role in many cardiovascular processes including myocardial infarction, reperfusion injury, angiogenesis, apoptosis, cardiac allograft rejection, and myocarditis. Radiolabeling leukocytes with 99mTc or 111In has been carried out in the past and requires removal of blood and in vitro labeling techniques prior to reinjection for imaging67;68. These techniques carry concern for nonspecific activation of the cells that may interfere with the localization in vivo. 67Ga-citrate has also been used but has been shown to be relatively nonspecific69. 18F-labeled deoxyglucose (FDG) PET imaging takes advantage of increased metabolic activity of inflammatory cells but can also be relatively nonspecific as a change in glucose uptake can be associated with other tissues and disease processes including tumors70;71. There is tremendous interest in developing more specific noninvasive imaging techniques to detect inflammation in myocardium.
Injury to myocytes in the setting of inflammation leads to the disruption of cellular membranes and the release of myosin heavy chain. In order to take advantage of this extracellular exposure of myosin in the setting of inflammation and necrosis, monoclonal antibody to myosin was generated in hopes of applying this for imaging. Early attempts to visualize myosin utilized 111In-labeled antimyosin antibodies to visualize myocyte damage in myocardial infarction72. Other studies utilized 99mTc-labeled monoclonal antibody fragments to quantitate the decree of myosin exposure in patients in the setting of acute myocardial infarction and correlate it with necrosis73. Inflammation associated with myocarditis, an inflammatory process not associated with ischemia, were also carried out using 111In-antimyosin antibodies74;75. Utilizing antimyosin antibody imaging, patients with dilated cardiomyopathy and lower ejection fractions revealed positive studies, suggesting a role for this for stratifying appropriate patients for cardiac transplantation. Though these initial studies showed promise, the background antibody binding to necrotic debris in the cell was high, and therefore the specificity of 111In-labeled antimyosin antibody turned out to be very low (25-50%)76.
Another monoclonal antibody against an extracellular matrix protein tenascin-C, which appears to be involved in wound healing and inflammation, has been identified as another potential imaging agent. In rodent models of myocarditis, 111In-labeled antitenescin-C localizes to the sites of myocardial inflammation77. Using a dual isotope SPECT approach with 111In-antitenescin-C and 99mTc-sestamibi, the antibody localized to the injured septal wall by in vivo imaging.
LTB4 is a lipid mediator synthesized from arachidonic acid and secreted by neutrophils, macrophages, and endothelial cells as a potent chemotactic agent78;79. The LTB4 receptor can be found on neutrophils and signaling through this receptor stimulates endothelial adhesion and superoxide production. Recently, a radiolabeled LTB4 receptor antagonist, 99mTc-RP517, was developed for in vivo imaging of inflammation 80;81. 99mTc-RP517 localized to sites of inflammation induced by S. aureas and E. coli infection, and chemical (phosphorbol-ester)-induced bowel inflammation.
When prepared with human peripheral whole blood in vitro, fluorinated RP517 localized to neutrophils by fluorescence activated cell sorter (FACs) analysis82. This confirmed the potential to label human blood neutrophils with 99mTc-labeled RP517. In an attempt to characterize the in vivo imaging ability of 99mTc-RP517, a canine model of post ischemic myocardial inflammation was utilized. 99mTc-RP517 was injected into open chest dogs before occlusion and reperfusion. There was an inverse relationship between radiotracer uptake and occlusion flow, suggesting localization of the imaging agent to the site of ischemic inflammation (Figure 4). Ex vivo segment analysis revealed that 99mTc-RP517 correlated with the neutrophil enzyme, myeloperoxidase. Intramyocardial injection of TNFα also correlated with 99mTc-RP517 uptake and concomitant myeloperoxidase activity, again supporting localization to the site of inflammation. One concern regarding the application of 99mTc-RP517 is the lipophilic nature of the molecule, resulting in high hepatobiliary clearance and thus large amounts of gastrointestinal uptake. To overcome this, alternative constructs of the LTB4 antagonist are currently being evaluated83.
Ventricular remodeling is a complex biological process that involve inflammation, repair, and healing with specific biochemical and structural alterations in the myocardial infarct and peri-infarct regions as well as remote regions84;85. The process is one of adaptation to form a scar that allows a degree of mechanical stability. The remodeling process involves several key cell types an structural elements, including myocardial cells, endothelial cells, inflammatory cells and the extracellular matrix.
Early in the first weeks after a myocardial infarction, an innate immune response initiates a complex process of wound healing in the necrotic tissue. This process evolves into a more chronic remodeling process that can involve hypertrophy, chamber dilation and, depending on the success of healing, heart failure86.
Matrix metalloproteinases (MMPs) are a family of zinc-containing enzymes that play a key role in ventricular remodeling by degradation of the extracellular matrix87;88. MMPs play an integral role in infarct expansion and left ventricular dilation. Gene deletion of MMPs has been demonstrated to have some cardioprotective effects from ventricular dilation and rupture post-infarct89. Pharmacologic inhibition of MMPs has also been shown to decrease left ventricular dilation in infarct models90-92.
Factor XIII has been shown to be crucial in organizing the new matrix of the scar by involvement with extracellular matrix turnover and regulation of inflammatory cascades93;94. Mice with decreased levels of factor XIII demonstrate increased ventricular dilation and post infarct rupture. Patients with infarct rupture were demonstrated to have lower levels of factor XIII in their myocardium. Factor XIII is activated by thrombin and often decreased in the setting of acute myocardial infarction in part because of therapeutic inhibition of thrombin. It is hypothesized that supplementing Factor XIII activity may have a beneficial role in post infarct remodeling.
A critical system that is locally activated during remodeling and contributes to the progression to heart failure is the rennin-angiotensin system95. As healing and remodeling are unsuccessful in the failing heart, there is increased expression of prorenin, renin, and angiotensin-converting enzyme (ACE). Activation of this system through signaling pathways mediated by the angiotensin II type I receptor (AT1) leads to myocyte hypertrophy, interstitial and perivascular collagen deposition, and myocyte apoptosis96. Inhibition of this pathway has been demonstrated to reverse the functional abnormalities associated with this negative remodeling.
By radiolabeling molecules that target MMPs like pharmacological inhibitors that specifically bind to the catalytic domain, MMP activation post infarct can be visualized in vivo (Figure 5)97. Initial studies involved non imaging techniques with 111In-labeled broad-spectrum MMP inhibitor (RP782), a molecule that selectively targets activated MMPs. This MMP targeted agent demonstrated a favorable biodistribution in a murine model of myocardial infarction. One week after myocardial infarction, 111In-RP782 localized primarily within the infarct region, although a lesser increase in retention was seen in the remote non-infarcted regions of the heart, consistent with global MMP activation and remodeling.
Further imaging studies were carried out utilizing 99mTc-labeled analog of RP782 (99mTc-RP805) and hybrid SPECT/CT imaging with a dual isotope protocol involving 99mTc-RP805 imaging and adjunctive 201Tl- perfusion imaging. The dual isotope imaging studies revealed MMP activation within the perfusion defect region. This suggests that MMP activation is taking place primarily within the sites of injury and is proof of concept that molecules that target MMP activation might be utilized to evaluate ventricular remodeling, and therapeutic interventions directed at inhibition of MMP activation.
111In-DOTA-FXIII is a radiolabeled glutaminase factor XIII substrate analogue that Factor XIII recognizes and cross-links to extracellular matrix proteins (Figure 6)94. 111In-DOTA-FXIII accumulates in areas of increased factor XIII activity. In a murine model of myocardial infarction, this 111In-labeled peptide substrate was demonstrated to be decreased in infarcts of animals treated with the direct thrombin inhibitor, dalteparin. Moreover, dalteparin treatment increased the risk of infarct rupture. Conversely, mice treated with factor XIII intravenously exhibited increased factor XIII activity in the infarct zone and demonstrated more rapid inflammatory turnover of neutrophils and increased recruitment of macrophages to the site of infarction. There was also increased collagen synthesis and capillary density in the factor XIII-treated animals, suggesting improved healing post infarction.
A number of ACE inhibitors and AT1 antagonists have been radiolabeled for molecular imaging techniques95. In a study of explanted hearts from patients with ischemic cardiomyopathy, 18F-fluorobenzoyl-lisinopril was used to assess ACE levels in infracted myocardium and fibrosed tissue98. The study demonstrated that the radiolabeled ACE inhibitor bound with some degree of specificity to areas adjacent to the infarct. Other studies using AT1 antagonists have demonstrated a differential between ACE activity and AT1 levels99. In an ovine model of heart failure, ACE activity was primarily in the vascular endothelium while AT1 was upregulated in the myofibroblasts of the infarct region. In a murine model of acute myocardial infarction, a 99mTc-labeled AT1 receptor peptide analogue was developed and demonstrated specificity to the myofibroblasts that localized to the infarct region in the weeks following the infarction. These early studies suggest that the neurohormonal changes that take place within an infarction may be utilized to identify those at risk for developing significant heart failure after myocardial infarction. Much more work is needed to assess the feasibility of these agents for imaging of post-infarction remodeling in clinical trials.
In summary, molecular imaging represents a targeted approach to non-invasively assess biological processes in vivo. The goal of molecular imaging is to develop an approach to studying not only the disease process but more importantly the efficacy of an individually tailored therapeutic regimen. The relatively high sensitivity of radiotracer-based imaging approaches such as SPECT and PET have been of great use in the practical application of molecular imaging techniques. Research has demonstrated the feasibility of specific targeted imaging approaches in the evaluation of angiogenesis, apoptosis, inflammation, and ventricular remodeling. Combining nuclear (SPECT or PET) and CT imaging modalities should help overcome issues of attenuation or partial volume effects and improve quantitative accuracy. The evolution of hybrid imaging systems and imaging protocols that include application of dual isotopes for monitoring physiological parameters (metabolism or perfusion) with targeted molecular probes shows promise in the areas of myocardial infarction and angiogenesis. Tailoring gene therapy with PET reporter constructs should allow for optimization of therapeutic efficacy in the areas of angiogenesis. The role for apoptotic imaging in understand reperfusion injury and the effects of therapeutic interventions, or in identifying the presence and severity of graft rejection following cardiac transplantation without the need for biopsy shows some promise. Imaging the activation of matrix metalloproteinases, active factor XIII or the levels of neurohormonal activation during ventricular remodeling may guide therapeutic regimens that could help positively influence outcomes post infarction. In conclusion, targeted radiotracer-based molecular imaging is clearly feasible and may play an important role in the evaluation and management of cardiovascular disease, including the future investigation of novel genetic or cell based therapeutic interventions.
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