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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptNIH Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Circ Cardiovasc Imaging. Author manuscript; available in PMC May 1, 2014.
Published in final edited form as:
PMCID: PMC3805276
NIHMSID: NIHMS473879

Fluorescence Tomography of Rapamycin-Induced Autophagy and Cardioprotection In Vivo

Howard H. Chen, PhD,1,2,3 Choukri Mekkaoui, PhD,1,3 Hoonsung Cho, PhD,3,4 Soeun Ngoy, BS,5 Brett Marinelli, BS,2,6 Peter Waterman, BS,2,6 Matthias Nahrendorf, MD, PhD,2,6 Ronglih Liao, PhD,5 Lee Josephson, PhD,1,3,4 and David E. Sosnovik, MD1,2,3

Abstract

Background

Autophagy is a biological process during which cells digest organelles in their cytoplasm and recycle the constituents. The impact of autophagy in the heart, however, remains unclear in part due to the inability to noninvasively image this process in living animals.

Methods and Results

Here, we report the use of fluorescence molecular tomography (FMT) and a cathepsin activatable fluorochrome to image autophagy in the heart in vivo following ischemia-reperfusion and rapamycin therapy. We show that cathepsin-B activity in the lysosome is upregulated by rapamycin and that this allows the expanded lysosomal compartment in autophagy to be imaged in vivo with FMT. We further demonstrate that the delivery of diagnostic nanoparticles to the lysosome by endocytosis is enhanced during autophagy. The upregulation of autophagy by rapamycin was associated with a 23% reduction (p<0.05) of apoptosis in the area-at-risk (AAR), and a 45% reduction in final infarct size (19.6 +/− 5.6% of AAR with rapamycin versus 35.9 +/− 9.1% of AAR without rapamycin, p<0.05).

Conclusions

The ability to perform noninvasive tomographic imaging of autophagy in the heart has the potential to provide valuable insights into the pathophysiology of autophagy, particularly its role in cardiomyocyte salvage. While additional data are needed, our study supports the investigation of rapamycin therapy in patients with acute coronary syndromes.

Keywords: autophagy, apoptosis, molecular imaging, rapamycin, myocardium

Autophagy, a biological process in which the cell digests a portion of its own cytoplasmic contents, is classically upregulated during starvation.1 The upregulation of autophagy, however, has also been documented in neurodegeneration,1 myocardial ischemia,2,3 and cardiomyopathy.4 It remains unclear, however, whether the upregulation of autophagy in the heart is protective or deleterious.5,6 It has been postulated by some that by clearing damaged mitochondria, which are a potent proapoptotic stimulus, that autophagy protects the cardiomyocyte (CM) from apoptotic cell death.1,7 Others have proposed that autophagy in itself may result in cell death.8 This uncertainty is driven in large part by the biological complexity of the process but also by the absence of a probe-based technique to image autophagy in vivo.

Autophagy, apoptosis and necrosis constitute a spectrum of processes involved in cell injury and death. While probe-based techniques have been developed to image apoptosis,911 and necrosis,12 in the heart in vivo, current techniques for determining the autophagic state of a cell are limited to genetic constructs and the use of fluorochromes with little ability to penetrate tissue.13 A strong need thus exists for a noninvasive technique supporting the in vivo detection of autophagy in the heart to be developed. Our goal here was to exploit the upregulation of cathepsin activity in the expanded lysosomal compartment during autophagy to develop such an approach. A model of ischemia-reperfusion (IR) plus rapamycin (RAP) treatment was used to create a robust model of CM autophagy in this proof-of-principle study. Rapamycin is a macrolide immunosuppressant known to induce autophagy via its inhibition of mTOR (mammalian target of rapamycin). Rapamycin and its analogues have also been used to treat transplant rejection and restenosis, and have an established safety profile for clinical use.1,7 A cathepsin-activatable near infrared fluorochrome (CAF-680) and fluorescence molecular tomography (FMT) of the heart in vivo were used to demonstrate the feasibility of the approach. We further aimed to determine whether the augmentation of CM autophagy by RAP during IR would be associated with a reduction in CM apoptosis and whether it would generate a protective or a deleterious effect.

Methods

Animal Protocol

The upregulation of autophagy in vivo was determined by the signal emitted from the cathepsin-activatable near infrared fluorochrome (CAF-680). As shown in Figure 1, four groups of mice were imaged: mice with ischemia-reperfusion injury (IR), mice with IR and postconditioning (IR+PC), mice with IR plus rapamycin treatment before coronary ligation (IR+preRAP) and mice with IR plus rapamycin treatment after coronary ligation (IR+postRAP). In all groups the CAF (ProSense-680, Perkin Elmer, Waltham MA) was injected at the onset of reperfusion and fluorescence imaging of the heart was performed 4 hours later. An initial cohort of mice was imaged ex vivo with fluorescence reflectance imaging (FRI) to establish proof-of-principle. In vivo imaging was subsequently performed in cohorts of mice with IR and IR+preRAP using fluorescence tomography (FMT) and micro-CT.

Figure 1
Molecular imaging of autophagy in the myocardium. (A) Four protocols were used: 1) Ischemia-reperfusion (IR) only, 2) Pretreatment with rapamycin followed by IR (IR+preRAP), 3) Treatment with rapamycin after the onset of coronary ligation (IR+postRAP), ...

IR injury was induced in female C57Bl6 mice by a 35-min ligation of the proximal left coronary artery (LCA), and was followed by 4 hours of reperfusion. Mice in the IR+preRAP group received rapamycin (3mg/kg body weight) by intraperitoneal injection 2 hours prior to the coronary ligation. Mice in the IR+postRAP group were injected intravenously with the identical dose of rapamycin during the period of coronary ligation. The postconditioning protocol involved 6 cycles of alternating ischemia (10sec) and reperfusion (10sec) before full reperfusion.14 Mice in the ex vivo imaging arms were injected with 2 nmol of CAF-680, while those in the in vivo imaging arm received 5 nmol. CAF-680 was injected intravenously via the tail vein at the onset of reperfusion. All surgical procedures were performed in accordance with animal protocols approved by the Institutional Animal Care and Use Committee of the Massachusetts General Hospital.

Fluorescence Reflectance Imaging (FRI)

The initial assessment of CAF-680 activity in the myocardium was performed with FRI. Mice with IR (n=7), IR+PC (n=7) and IR+preRAP (n=7) were studied. The mice were euthanized 4 hours after reperfusion and the heart was cut in the short axis plane into apical, midventricular and basal sections. FRI of these sections was performed on a commercial imaging system (Kodak In-Vivo FX Pro, Carestream Health, Rochester NY) with a spatial resolution of 48 μm, a 30 sec exposure, and wavelengths of 650 nm (excitation) and 700 nm (emission).

FRI was also performed in a separate cohort of mice with IR (n=7), IR+preRAP (n=7) and IR+postRAP (n=7) that were injected with fluorescently labeled Annexin-V (ANX-750) and fluorescent microspheres. The fluorescent microspheres (10 μm diameter, Life Technologies, Carlsbad CA) were given by intracardiac injection 5 min prior to reperfusion. The ANX-750 probe (Annexin Vivo 750, Perkin Elmer) was injected at the onset of reperfusion. The hearts were sectioned, as described above, 4 hours after reperfusion and FRI was performed at 750nm excitation/800nm emission for Annexin-V and 550nm(ex)/600nm(em) for the fluorescent microspheres.

A third cohort of mice with IR (n=7), IR+preRAP (n=7) and IR+postRAP (n=7) was used to image infarct size at 24 hours by TTC (2,3,5-triphenyltetrazolium chloride, GFS Chemicals, Powell OH). Fluorescent microspheres were injected as described above. The mice were euthanized 24 hours after reperfusion and the heart sectioned in its short axis into 1mm thick slices. The distribution of the microspheres was imaged on a multispectral imaging platform (Kodak In-Vivo FX Pro, Carestream) and images of TTC were obtained with a flatbed scanner (Hewlett Packard, Palo Alto CA).

Image analysis was performed in ImageJ (NIH, Bethesda MD). The average fluorescence signal in the ischemic area of the myocardium (defined in those hearts not injected with microspheres as the region between the upper and lower right ventricular insertion points) was compared to that in the non-ischemic septum (Figure 1B). The contrast to noise ratio (CNR) between these two zones was calculated as: CNR= (Signalischemic – Signalnon-ischemic)/standard deviationnoise.15 Automated segmentation of the Annexin-V and TTC images was performed in ImageJ by using a signal intensity threshold greater than 2 standard deviations above that in the non-ischemic septum. The AAR in the fluorescent microsphere images was segmented manually.

In vivo Imaging

FMT-CT was performed in mice with IR+preRAP (n=7) and IR (n=7) four hours after reperfusion and the intravenous injection of 5 nmol of CAF-680. CT imaging was performed on a commercial Micro PET-CT system (see supplement). The mice were transferred from the CT to the FMT system while still anesthetized and held in position in a plexiglass holder.

In vivo FMT was performed with the 680/700 nm excitation/emission filter setting on a commercial FMT system (Perkin Elmer). 26 frontal slices of 0.5 mm thickness in the z-direction, with an in-plane resolution of 1 × 1 mm2, were acquired. Data were post-processed with a normalized Born forward equation to reconstruct 3-dimensional fluorochrome concentration distribution maps for quantification, as described previously.15 Fusion of 3D fluorescence and CT datasets was performed offline using the fiducial landmarks on the frame of the plexiglass holder (see supplement).

Microscopy

Following FMT, to determine the cellular and subcellular locations of CAF-680, the hearts of the IR+preRAP mice were excised and sectioned for confocal microscopy. Immunofluorescence staining of the lysosomal marker LAMP-2 was performed with a PE-conjugate antibody (SC-19991PE anti-mouse, Santa Cruz

Biotechnology, Santa Cruz CA) according to manufacturer’s instructions (see supplement). Subsequently CAF-680 fluorescence was excited with the 633 nm Helium-Neon laser and acquired with the 650 nm line-pass filter. Differential Interference Contrast (DIC) images were acquired as well to visualize cellular anatomy. The non-ischemic septum and the ischemic area of injury were defined by the RV insertion points. Fluorescence in the ischemic region was compared to that in the non-ischemic septum in two ways: 1) overall fluorescence intensity in each region normalized to area and, 2) the number of hyperintense fluorescence punctates in each zone normalized to area. Colocalization of the CAF-680 and LAMP-2 signals was quantified using ImageJ.

In Vitro Studies

To further elucidate the mechanism of the enhanced CAF-680 activation by rapamycin, a series of experiments were performed on CHO and H9C2 cells (ATCC, Manassas VA). Cathepsin activity in control cells, and cells exposed to rapamycin, was determined using a commercial fluorometric assay (see supplement).

To determine the effects of rapamycin on endocytosis, the cells were incubated with FER-680, which is synthesized by attaching the fluorochrome CyAl5.5 to the Feraheme (Ferumoxytol, AMAG Pharmaceuticals, Lexington MA) nanoparticle. Feraheme has a blood half-life in humans of over 10 hours and is only very slowly internalized by endocytosis in normal cells.16 The internalization of FER-680 was determined by FACS (LSRII, BD Biosciences, Franklin Lakes, NJ) and further by costaining (50 nM, 1 minute incubation) the cells with the lysosomal marker LysoTracker Red (Life Technologies).

Impact on Infarct Size

Two strategies were employed to investigate whether the induction of autophagy in the IR+preRAP (n=14) and IR+postRAP (n=14) mice would exert a protective or deleterious effect on infarct size. Fluorescently labeled Annexin-V (ANX-750) was injected intravenously at the onset of reperfusion and used to determine the extent of cell injury by FRI 4 hours after injection. In addition, TTC staining was used to determine infarct size 24 hours after IR. The area of ANX-750 uptake and infarct size by TTC where both normalized to the area-at-risk (AAR), determined by the injection of fluorescently labeled microspheres.

Statistics

One-way ANOVA with a Tukey post-test was carried out to compare the IR, IR+preRAP, IR+postRAP and IR+PC groups. An unpaired t-test with a 2-tailed p-value was applied to compare the in vivo FMT data, and all other comparisons between 2 groups. All tests were performed in Prizm (Graphpad, La Jolla, CA) and results reported as mean ± SEM. A p- value of <0.05 was needed to meet significance.

Results

FRI of the mice subject to IR and IR+PC showed that fluorescence activity in the hearts of these mice was not increased (Figure 1). However, a significant increase in fluorescence activity was seen in the mice with IR pretreated with rapamycin (IR+preRAP). The contrast to noise ratio (CNR) between the ischemic lateral wall and the non-ischemic septum was 8.8 +/− 2.0 in the IR+preRAP mice versus 0.65 +/− 2.0 in the IR mice (p<0.05). The activation of CAF-680 was thus both highly sensitive and specific for the augmentation of CM autophagy in IR by rapamycin.

The activation of CAF-680 in the RAP treated mice could also be robustly imaged in vivo. For logistical reasons in vivo imaging was performed in the IR and IR+preRAP groups. The presence of fiducial markers in the animal cradle allowed the 3D CT and FMT datasets to be accurately coregistered (Figure 2). Volume rendering of the heart allowed fluorescence arising in the heart and liver to be definitively distinguished. Hepatic activation of CAF-680, which is a normal finding, was seen in both the IR and IR+preRAP mice imaged with FMT. CAF-680 activation in the heart, however, was seen only in the IR+preRAP mice (Figure 2).

Figure 2
Fused FMT-CT of cathepsin activity in the heart in vivo. (A, B) The infusion of iodinated contrast allowed the cardiac chambers, aorta and kidneys to be segmented on the micro-CT images. Volume rendering of the outer surface of the heart (red mesh) was ...

CAF activation in the myocardium of the IR+preRAP mice could be successfully imaged in vivo in a 3D and depth-resolved manner (Figure 3). Masking the fluorescence signal in the liver, which was segmented anatomically in the CT images, was performed to optimize dynamic range.

Figure 3
Noninvasive tomographic imaging of autophagy in the heart in vivo. (A–D) FMT-CT images of a mouse following IR and rapamycin pretreatment (IR+preRAP). Hepatic fluorescence has been masked out. (A) Left lateral view of the heart and skeleton. (B) ...

Fluorescence activity was greatest in the anterior and lateral walls of the apical half on the left ventricle (Figure 3), consistent with the location of the ischemic injury. Total fluorescence activity in the hearts was summed and was significantly greater in the mice with IR+preRAP than IR alone (21.0 +/− 5.6 versus 0.78 +/− 0.4, p=0.004).

Confocal microscopy confirmed that the activation of CAF-680 was due to CM autophagy. At low magnification (Figure 4A) numerous punctate foci of CAF-680 activation were seen in the at-risk anterior and lateral walls of the myocardium, but not in the septum (S). Under high magnification, the localization of these fluorescence foci within the CMs in the ischemic zone could be confirmed (Figure 4B). Both the total fluorescence intensity and the number of fluorescent foci were significantly higher in the ischemic myocardium than in the non-ischemic septum (Figure 4C). Staining of the slides with a fluorescent antibody to the lysosomal marker LAMP-2 showed that 79.0 +/− 5.8% of the CAF-680 signal arose from within lysosomes (Figure 4D–F).

Figure 4
Confocal microscopy of CAF-680 activation in autophagic cardiomyocytes. Hearts from mice (n=7) with IR+preRAP were sectioned after FMT. (A) CAF-680 activation produces fluorescent punctates in the area of injury. The septum (S) is injury-free and is devoid ...

Exposure of CHO and H9C2 cells to rapamycin significantly increased cathepsin-B activity (measured by direct enzymatic assay) in these cells (Figure 5A). As shown in Figure 5B, rapamycin also significantly increased the uptake of FER-680 in the cell lines examined. Colocalization of FER-680 and LysoTracker Red (LTR) showed that the internalized nanoparticles were trafficked to the lysosomal compartment (Figure 5C).

Figure 5
Mechanism of CAF-680 activation by autophagic cells. (A) A marked increase in lysosomal cathepsin-B activity was seen in CHO and H9C2 cells exposed to rapamycin (p<0.01 versus unexposed controls). (B) Endocytosis of the non-targeted fluorescent ...

The induction of autophagy by RAP produced a marked cardioprotective effect (Figure 6). RAP pretreatment decreased the portion of the AAR positive for Annexin-V from 90.4 +/− 6.9% to 74.8 +/− 8.0% (p<0.01). The intravenous injection of RAP post coronary ligation produced a similar reduction in the Annexin-V positive area (69.3 +/− 5.9%, p<0.001 versus control). Infarct size at 24 hours by TTC staining was 35.9 +/− 9.0% in the IR mice and decreased to 24.1 +/−7.3% in the IR+preRAP mice (p<0.05) and 19.6 +/− 5.6% in the IR+postRAP mice (p<0.01).

Figure 6
Cardioprotective effect of rapamycin-induced autophagy in ischemia-reperfusion injury. (A) Ex vivo fluorescence reflectance images of mice injected with fluorescent microspheres and an annexin-labeled fluorochrome are shown. The absence of fluorescent ...

Discussion

Imaging of Autophagy

Autophagy is a complex biological process and requires the execution of an elaborate pathway of signals and steps.1,8 However, despite the sophisticated biological understanding of the process, probe chemistries and whole-animal imaging methodologies to image autophagy have not been developed. Here we show that the upregulation of cathepsin activity in the expanded lysosomal space allows CM autophagy to be imaged in vivo with an activatable near infrared fluorochrome. We further show that the upregulation of CM autophagy by rapamycin is associated with a significant reduction in CM apoptosis and a marked cardioprotective effect.

Our broad goal of developing a probe based tomographic technique to image autophagy in vivo required three key elements: the use of a cathepsin-activatable near infrared fluorochrome, the use of the advanced reconstruction algorithms inherent in FMT,17 and the ability to separate autophagy-mediated activation of CAF-680 from that due to infiltrating leukocytes.18 Neutrophil infiltration in ischemic myocardium occurs 4–6 hours post injury, while macrophage infiltration occurs between 12–24 hours.19 Here, by imaging CAF-680 activity within 4 hours of injury, the potential for immune-mediated probe activation was minimized. Two additional findings suggest that the CAF-680 activation seen in the myocardium at 4 hours was not immune mediated.

Confocal microscopy showed that the bulk of the CAF-680 signal was localized within the lysosomes of CMs in the ischemic zone. Furthermore, CAF-680 activation was dramatically enhanced by rapamycin pretreatment, which exerts a significant anti-inflammatory effect. In fact, the exposure of neutrophils to rapamycin has been shown to attenuate their migration and chemotaxis.20,21 Taken together, these factors strongly suggest that the activation of CAF-680 within 4 hours of IR+RAP was a specific marker for the upregulation of CM autophagy.

Impact of Rapamycin in Ischemic Injury

CM death within the first few hours of IR is mediated predominantly by apoptosis.22,23 Interestingly, we show here that although annexin-positivity was detected in almost the entire area-at-risk (AAR) within the first few hours of IR, only a fraction of the AAR became TTC positive at 24 hours. This result is consistent with our previous finding that the majority of annexin-positive myocardium (by MRI) does not show evidence of late gadolinium enhancement (cell necrosis) 4 hours after IR.9 These results suggest that not all annexin-positive cells undergo irreversible cell death and that the expression of phosphatidylserine on the outer CM membrane is a partially reversible phenomenon.

The ability of phosphatidylserine positive myocardium to remain viable has also been demonstrated by Kenis et al, who found after brief ischemia that the expression of phosphatidylserine on the outer cell membrane of CMs was cyclic and did not correlate with histological evidence of apoptosis or necrosis.23 The duration of ischemia (35 min) used in our study was significantly longer and, likewise, showed that annexin uptake did not automatically correlate with CM death. We have also previously shown that the stabilization of the apoptotic cell membrane with an annexin-labeled nanoparticle attenuates cell rupture and death.24 A certain threshold of injury may thus be needed for an annexin positive cell to fully execute the cell death cascade.25 Further study, however, will be needed to fully elucidate the fate of annexin-positive cells.

Rapamycin treatment was associated with a significant increase in CM autophagy, and significant reductions in CM apoptosis and infarct size. These data suggest that rapamycin-induced autophagy in IR exerts a strong protective effect. It is possible that the impact of rapamycin on the PI3-kinase/AKT pathway and on neutrophil migration may also play a role in its cardioprotective effect.20,21,26 However, given the dramatic rise in CAF-680 activation produced by rapamycin, it is likely that these effects are secondary to the protective effect of markedly enhanced autophagy.

The digestion of dysfunctional cytoplasmic organelles in autophagy is well described.1 This process provides vital nutrients and energy to an injured or starving cell. Here we show that rapamycin-induced autophagy also causes cells to increase the rate at which they endocytose nutrient containing materials, such as dextran-containing nanoparticles, from the extracellular space.27 This likely further enhances the cells’ ability to maintain energy homeostasis in ischemic injury, and may aid CM survival.

Postconditioning has been shown to exert a cardioprotective effect in both animal models and humans.28,29 We show here, however, that this effect is not mediated via increased autophagy. CAF-680 activation in the mice exposed to postconditioning was no higher than in those with conventional IR. This suggests that the ability of postconditioning to maintain mitochondrial integrity is sufficient to negate the need for the clearance of damaged mitochondria by autophagy. Preconditioning has also been shown to be cardioprotective,30 and may exert similar effects on the CM to those of rapamycin pretreatment.26 This raises the possibility that the protective effect of preconditioning may in part be mediated by increased autophagy.

Approaches to Image Autophagy

Genetically engineered animals and cells that overexpress a fluorescent reporter protein construct are widely used to study autophagy.5,13,27 This approach allows the signaling and molecular mechanisms involved in autophagy to be accurately elucidated.5 However, fluorochromes in the visible spectrum suffer from low tissue penetrance, high attenuation and high background autofluorescence. These genetic reporter approaches thus are unsuited to quantitative or depth resolved tomographic imaging of autophagy.17

While the approach presented here represents a significant advance in the imaging of autophagy, its potential for clinical translation is limited. The development of a cathepsin-activated drug and a device for fluorescence tomography in humans are both formidable challenges. The development of positron emission tomography (PET) detectable agents to image autophagy in vivo will thus be of utmost importance. This not only offers a route towards clinical translation but also the potential to increase diagnostic sensitivity. The fluorescent approach described here, however, is of major preclinical value, and has the potential to provide important mechanistic insights and facilitate the development of novel pharmaceuticals.

Conclusion

The marked cardioprotective effect produced by rapamycin in this study is in agreement with prior observations in rats.31 The mice in the IR+postRAP cohort in this study were injected intravenously with rapamycin after the onset of myocardial ischemia and experienced a 45% reduction in infarct size. This raises the possibility of using rapamycin to reduce infarct size in patients presenting with acute coronary syndromes. The molecular mechanisms underlying the cardioprotective effect of rapamycin will need to be more fully elucidated. Nevertheless, the demonstration that a well-tolerated and clinically-approved agent has the potential to exert a marked cardioprotective effect is highly noteworthy.

In conclusion, we have shown here for the first time that quantitative tomographic imaging of autophagy can be performed in vivo. This has broad implications not only for cardiovascular disease, but also for neurodegenerative disease and cancer. Serial in vivo imaging of the same animal will allow the natural history of autophagy and its pathophysiological impact to be better understood. Furthermore, our data suggest that the induction of autophagy by rapamycin exerts a powerful cardioprotective effect. This strategy is highly translatable and could be of significant utility in reducing the potential of acute coronary syndromes to result in chronic heart failure.

Cardiomyocyte (CM) death, even in the presence of adequate reperfusion, remains a major clinical problem. Recently, a new response to ischemic stress, termed autophagy, has been described. It remains unclear, however, whether autophagy in the heart is protective or deleterious. The aims of this study were thus: 1) To develop a mechanism to image autophagy in vivo to enhance the understanding of this process, and 2) to investigate whether the induction of autophagy with rapamycin during ischemia-reperfusion would prove protective or deleterious. The imaging of autophagy was performed by exploiting the upregulation of lysosomal cathepsins during this process. Fluorescence imaging of a cathepsin-activatable fluorochrome was performed. In the mice injected with rapamycin, a large increase in the fluorescence signal was seen in the injured myocardium. This was not seen in the absence of rapamycin injection. The injection of rapamycin was associated with a significant reduction of apoptosis in the area-at-risk, as well as a reduction in final infarct size. This protective effect was seen even when the rapamycin was injected after coronary ligation. Further study is needed to determine whether the protective effects of rapamycin/autophagy are durable and are seen in large animal models as well.

Supplementary Material

Acknowledgments

We would like to thank the Ragon Institute flow cytometry and microscopy core for their technical support.

Sources of Funding

This work was funded in part by the following grants from the NIH (R01 HL093038 (DES), R01 EB011996 (LJ) and P41RR14075 (Martinos Center).

Footnotes

Disclosures

None.

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