Ian Y. Chen

Molecular Imaging of Cardiac Cell Transplantation in Living Animals Using Optical Bioluminescence and Positron Emission Tomography

Background—The current method of analyzing myocardial cell transplantation relies on postmortem histology. We sought to demonstrate the feasibility of monitoring transplanted cell survival in living animals using molecular imaging techniques. Methods and Results—For optical bioluminescence charged-coupled device imaging, rats (n=20) underwent intramyocardial injection of embryonic rat H9c2 cardiomyoblasts (3×106 to 5×105) expressing firefly luciferase (Fluc) reporter gene. Cardiac bioluminescence signals were present for more than 2 weeks with 3×106 cells: day 1 (627 000±15%), day 2 (346 100±21%), day 4 (112 800±20%), day 8 (78 860±24%), day 12 (67 780±12%), and day 16 (62 200±5% p · s−1 · cm2−1 · sr−1). For micro–positron emission tomography imaging, rats (n=20) received cardiomyoblasts (3×106) expressing mutant herpes simplex type 1 thymidine kinase (HSV1-sr39tk) reporter gene. Detailed tomography of transplanted cells is shown by 9-(4-[18F]-fluoro-3hydroxymethylbutyl)guanine ([18F]-FHBG) reporter probe and nitrogen-13 ammonia ([13N]-NH3) perfusion images. Within the transplanted region, there was a 4.48±0.71-fold increase of in vivo [18F]-FHBG activity and a 4.01±0.51-fold increase of ex vivo gamma counting compared with control animals. Finally, the in vivo images of cell survival were confirmed by ex vivo autoradiography, histology, immunohistochemistry, and reporter protein assays. Conclusions—The location(s), magnitude, and survival duration of embryonic cardiomyoblasts were monitored noninvasively. With further development, molecular imaging studies should add critical insights into cardiac cell transplantation biology.

US Imaging of Tumor Angiogenesis with Microbubbles Targeted to Vascular Endothelial Growth Factor Receptor Type 2 in Mice

PURPOSE To prospectively evaluate contrast material-enhanced ultrasonography (US) with microbubbles targeted to vascular endothelial growth factor receptor type 2 (VEGFR2) for imaging tumor angiogenesis in two murine tumor models. MATERIALS AND METHODS Animal protocols were approved by the Institutional Administrative Panel on Laboratory Animal Care. A US contrast agent, consisting of encapsulated gaseous microbubbles, was developed specifically to bind to VEGFR2 (by using anti-VEGFR2 antibodies and biotin-streptavidin interaction) which is up-regulated on endothelial cells of tumor blood vessels. VEGFR2-targeted microbubbles (MB(V)), control microbubbles (MB(C)), and nonlabeled microbubbles (MB(N)) were tested for binding specificity on cells expressing VEGFR2 (mouse angiosarcoma SVR cells) and control cells (mouse skeletal myoblast C2C12 cells). Expression of mouse VEGFR2 in culture cells was tested with immunocytochemical and Western blot analysis. Contrast-enhanced US imaging with MB(V) and MB(C) was performed in 28 tumor-bearing nude mice (mouse angiosarcoma, n = 18; rat malignant glioma, n = 10). Differences were calculated by using analysis of variance. RESULTS In cell culture, adherence of MB(V) on SVR cells (2.1 microbubbles per SVR cell) was significantly higher than adherence of control microbubbles (0.01-0.10 microbubble per SVR cell; P < .001) and significantly more MB(V) attached to SVR cells than to C2C12 cells (0.15 microbubble per C2C12 cell; P < .001). In vivo, contrast-enhanced US imaging showed significantly higher average video intensity when using MB(V) compared with MB(C) for angiosarcoma and malignant glioma tumors (P < .001). Results of immunohistochemical analysis confirmed VEGFR2 expression on vascular endothelial cells of both tumor types. CONCLUSION US imaging with contrast microbubbles targeted to VEGFR2 allows noninvasive visualization of VEGFR2 expression in tumor vessels in mice.

Collagen Matrices Enhance Survival of Transplanted Cardiomyoblasts and Contribute to Functional Improvement of Ischemic Rat Hearts

Background— Cardiac cell transplantation is limited by poor graft viability. We aimed to enhance the survival of transplanted cardiomyoblasts using growth factor-supplemented collagen matrices. Methods and Results— H9c2 cardiomyoblasts were lentivirally transduced to express firefly luciferase and green fluorescent protein (GFP). Lewis rats underwent ligation of the left anterior descending artery (LAD) ligation to induce an anterior wall myocardial infarction. Hearts (n=9/group) were harvested and restored ex vivo with 1×106 genetically labeled H9c2 cells either in (1) saline-suspension, or seeded onto (2) collagen-matrix (Gelfoam [GF];), (3) GF/Matrigel (GF/MG), (4) GF/MG/VEGF (10 &mgr;g/mL), or (5) GF/MG/FGF (10 &mgr;g/mL). Hearts were then abdominally transplanted into syngeneic recipients (working heart model). Controls (n=6/group) underwent infarction followed by GF implantation or saline injection. Cell survival was evaluated using optical bioluminescence on days 1, 5, 8, 14, and 28 postoperatively. At 4 weeks, fractional shortening and ejection fraction were determined using echocardiography and magnetic resonance imaging, respectively. Graft characteristics were assessed by immunohistology. Bioluminescence signals on days 5, 8, and 14 were higher for GF-based grafts compared with plain H9c2 injections (P<0.03). Signals were higher for GF/MG grafts compared with GF alone (P<0.02). GFP-positive, spindle-shaped H9c2 cells were found integrated in the infarct border zones at day 28. Left ventricular (LV) function of hearts implanted with collagen-based grafts was better compared with controls (P<0.05). Vascular endothelial growth factor or fibroblast growth factor did not further improve graft survival or heart function. Conclusions— Collagen matrices enhance early survival of H9c2 cardiomyoblasts after transplantation into ischemic hearts and lead to improved LV function. Further optimization of the graft design should make restoration of large myocardial infarctions by tissue engineering approaches effective.

Cardiovascular Molecular Imaging Focus on Clinical Translation

The past few decades have seen an explosion in the knowledge of the molecular basis of cardiovascular diseases owing to rapid advances in molecular biology research. An improved understanding of disease pathogenesis at the genomic, transcriptional, and proteomic levels has led to the discovery of promising experimental strategies for the prevention, diagnosis, and treatment of cardiovascular disease. Unfortunately, only a minute number of these strategies have survived the rigors of preclinical and clinical trials to become therapeutically useful. Furthermore, even these successful strategies must endure a prolonged process of translation from bench to bedside, partially owing to the lack of tools to directly interrogate the molecular events in patients. The strong impetus to develop noninvasive imaging techniques to visualize molecular changes in patients has given birth to the field of molecular imaging.1,2 Molecular imaging has its roots in nuclear medicine, in which radiolabeled imaging probes are injected into living subjects to assess the functionality of different organ systems. Unlike conventional diagnostic imaging techniques (eg, radiography and computed tomography [CT]) that delineate the anatomy of the cardiovascular system (eg, coronary luminal diameter), molecular imaging techniques have been designed and validated to study much smaller-scale molecular events (eg, gene expression) that may underlie disease processes. The complexity of molecular imaging lies in the requirement for molecular targeting, the design of which requires a solid understanding of the pharmacokinetics of the imaging probe and how it interacts with the molecular target. When the target is proven to be a biomarker, molecular imaging becomes a valuable tool for detecting disease before its clinical manifestation, stratifying disease severity, predicting disease progression, monitoring treatment efficacy, and prognosticating disease. These challenges must be met before the true potential of personalized medicine can be fully realized.3 Significant advances have been made in molecular imaging …

Imaging of VEGF receptor in a rat myocardial infarction model using PET.

Myocardial infarction (MI) leads to left ventricular (LV) remodeling, which leads to the activation of growth factors such as vascular endothelial growth factor (VEGF). However, the kinetics of a growth factors receptor expression, such as VEGF, in the living subject has not yet been described. We have developed a PET tracer (64Cu-DOTA-VEGF121 [DOTA is 1,4,7,10-tetraazadodecane-N,N′,N″,N‴-tetraacetic acid]) to image VEGF receptor (VEGFR) expression after MI in the living subject. Methods: In Sprague–Dawley rats, MI was induced by ligation of the left coronary artery and confirmed by ultrasound (n = 8). To image and study the kinetics of VEGFRs, 64Cu-DOTA-VEGF121 PET scans were performed before MI induction (baseline) and on days 3, 10, 17, and 24 after MI. Sham-operated animals served as controls (n = 3). Results: Myocardial origin of the 64Cu-DOTA-VEGF121 signal was confirmed by CT coregistration and autoradiography. VEGFR specificity of the 64Cu-DOTA-VEGF121 probe was confirmed by in vivo use of a 64Cu-DOTA-VEGFmutant. Baseline myocardial uptake of 64Cu-DOTA-VEGF121 was minimal (0.30 ± 0.07 %ID/g [percentage injected dose per gram of tissue]); it increased significantly after MI (day 3, 0.97 ± 0.05 %ID/g; P < 0.05 vs. baseline) and remained elevated for 2 wk (up to day 17 after MI), after which time it returned to baseline levels. Conclusion: We demonstrate the feasibility of imaging VEGFRs in the myocardium. In summary, we imaged and described the kinetics of 64Cu-DOTA-VEGF121 uptake in a rat model of MI. Studies such as the one presented here will likely play a major role when studying pathophysiology and assessing therapies in different animal models of disease and, potentially, in patients.

Micro–Positron Emission Tomography Imaging of Cardiac Gene Expression in Rats Using Bicistronic Adenoviral Vector-Mediated Gene Delivery

Background—We have previously validated the use of micro-positron emission tomography (microPET) for monitoring the expression of a single PET reporter gene in rat myocardium. We now report the use of a bicistronic adenoviral vector (Ad-CMV-D2R80a-IRES-HSV1-sr39tk) for linking the expression of 2 PET reporter genes, a mutant rat dopamine type 2 receptor (D2R80a) and a mutant herpes simplex virus type 1 thymidine kinase (HSV1-sr39tk), with the aid of an internal ribosomal entry site (IRES). Methods and Results—Rat H9c2 cardiomyoblasts transduced with increasing titers of Ad-CMV-D2R80a-IRES-HSV1-sr39tk (0 to 2.5×108 pfu) were assayed 48 hours later for reporter protein activities, which were found to correlate well with viral titer (r2=0.96, P <0.001 for D2R80A; r2=0.98, P <0.001 for HSV1-sr39TK) and each other (r2=0.97; P <0.001). Experimental (n=8) and control (n=6) athymic rats underwent intramyocardial injection of up to 2×109 pfu of Ad-CMV-D2R80a-IRES-HSV1-sr39tk and saline, respectively. Forty-eight hours later and weekly thereafter, rats were assessed for D2R80a-dependent myocardial accumulation of 3-(2-[18F]fluoroethyl)spiperone ([18F]-FESP) and HSV1-sr39tk–dependent sequestration of 9-(4-[18F]fluoro-3-hydroxymethylbutyl)guanine ([18F]-FHBG) using microPET. Longitudinal [18F]-FESP and [18F]-FHBG imaging of experimental rats revealed a good correlation between the cardiac expressions of the 2 PET reporter genes (r2=0.73; P <0.001). The location of adenovirus-mediated transgene expression, as inferred from microPET images, was confirmed by ex vivo gamma counting of explanted heart. Conclusions—The IRES-based bicistronic adenoviral vector can potentially be used in conjunction with PET for indirect imaging of therapeutic gene expression by replacing 1 of the 2 PET reporter genes with a therapeutic gene of choice.

Adenoviral Human BCL-2 Transgene Expression Attenuates Early Donor Cell Death After Cardiomyoblast Transplantation Into Ischemic Rat Hearts

Background— Cell transplantation for myocardial repair is limited by early cell death. Gene therapy with human Bcl-2 (hBcl-2) has been shown to attenuate apoptosis in the experimental setting. Therefore, we studied the potential benefit of hBcl-2 transgene expression on the survival of cardiomyoblast grafts in ischemic rat hearts. Methods and Results— H9c2 rat cardiomyoblasts were genetically modified to express both firefly luciferase and green fluorescent protein (mH9c2). The cells were then transduced with adenovirus carrying hBcl-2 (AdCMVhBcl-2/mH9c2). Lewis rats underwent ligation of the left anterior descending artery (LAD) to induce a sizable left ventricular (LV) infarct. Hearts were explanted and the infarcted region was restored using collagen matrix (CM) seeded with 1×106 mH9c2 cells (n=9) or AdCMVhBcl-2/mH9c2 cells (n=9). Control animals received CM alone (n=6) or no infarct (n=6). Restored hearts were transplanted into the abdomen of syngeneic recipients in a “working heart” model. Cell survival was evaluated using optical bioluminescence imaging on days 1, 5, 8, 14, and 28 after surgery. The left heart function was assessed 4 weeks postoperatively using echocardiography and magnetic resonance imaging. During 4 weeks after surgery, the optical imaging signal for the AdCMVhBCL2/mH9c2 group was significantly (P<0.05) higher than that of the mH9c2-control group. Both grafts led to better fractional shortening (AdCMVhBcl-2/mH9c2: 0.21±0.03; mH9c2: 0.21±0.04; control: 0.15±0.03; P=0.04) and ejection fraction (AdCMVhBcl-2/mH9c2: 47.0±6.2; mH9c2: 48.7±6.1; control: 34.3±6.0; P=0.02) compared with controls. Importantly, no malignant cells were found in postmortem histology. Conclusion— Transduction of mH9c2 cardiomyoblasts with AdCMVhBcl-2 increased graft survival in ischemic rat myocardium without causing malignancies. Both AdCMVhBcl-2/mH9c2 and mH9c2 grafts improved LV function.

Monitoring of the Biological Response to Murine Hindlimb Ischemia With 64Cu-Labeled Vascular Endothelial Growth Factor-121 Positron Emission Tomography

Background— Vascular endothelial growth factor-121 (VEGF121), an angiogenic protein secreted in response to hypoxic stress, binds to VEGF receptors (VEGFRs) overexpressed on vessels of ischemic tissue. The purpose of this study was to evaluate 64Cu-VEGF121 positron emission tomography for noninvasive spatial, temporal, and quantitative monitoring of VEGFR2 expression in a murine model of hindlimb ischemia with and without treadmill exercise training. Methods and Results— 64Cu-labeled VEGF121 and a VEGF mutant were tested for VEGFR2 binding specificity in cell culture. Mice (n=58) underwent unilateral ligation of the femoral artery, and postoperative tissue ischemia was assessed with laser Doppler imaging. Longitudinal VEGFR2 expression in exercised and nonexercised mice was quantified with 64Cu-VEGF121 positron emission tomography at postoperative day 8, 15, 22, and 29 and correlated with postmortem &ggr;-counting. Hindlimbs were excised for immunohistochemistry, Western blotting, and microvessel density measurements. Compared with the VEGF mutant, VEGF121 showed specific binding to VEGFR2. Perfusion in ischemic hindlimbs fell to 9% of contralateral hindlimb on postoperative day 1 and recovered to 82% on day 29. 64Cu-VEGF121 uptake in ischemic hindlimbs increased significantly (P<0.001) from a control level of 0.61±0.17% ID/g (percentage of injected dose per gram) to 1.62±0.35% ID/g at postoperative day 8, gradually decreased over the following 3 weeks (0.59±0.14% ID/g at day 29), and correlated with &ggr;-counting (R2=0.99). Compared with nonexercised mice, 64Cu-VEGF121 uptake was increased significantly (P≤0.0001) in exercised mice (at day 15, 22, and 29) and correlated with VEGFR2 levels as obtained by Western blotting (R2=0.76). Ischemic hindlimb tissue stained positively for VEGFR2. In exercised mice, microvessel density was increased significantly (P<0.001) compared with nonexercised mice. Conclusions— 64Cu-VEGF121 positron emission tomography allows longitudinal spatial and quantitative monitoring of VEGFR2 expression in murine hindlimb ischemia and indirectly visualizes enhanced angiogenesis stimulated by treadmill exercise training.

Detection of colonic polyps in a phantom model : Implications for virtual colonoscopy data acquisition

PURPOSE Virtual colonoscopy is a new method of colon examination in which computer-aided 3D visualization of spiral CT simulates fiberoptic colonoscopy. We used a colon phantom containing various-sized spheres to determine the influence of CT acquisition parameters on lesion detectability and sizing. METHOD Spherical plastic beads with diameters of 2.5, 4, 6, 8 and 10 mm were randomly attached to the inner wall of segments of plastic tubing. Groups of three sealed tubes were scanned at 3/1, 3/2, 5/1 collimation (mm)/pitch settings in orientations perpendicular and parallel to the scanner gantry. For each acquisition, image sets were reconstructed at intervals from 0.5 to 5.0 mm. Two blinded reviewers assessed transverse cross-sections of the phantoms for bead detection, using source CT images for images for acquisitions obtained with the tubes oriented perpendicular to the gantry and using orthogonal reformatted images for scans oriented parallel to the gantry. RESULTS Detection of beads of > or = 4 mm was 100% for both tube orientations and for all collimator/pitch settings and reconstruction intervals. For the 2.5 mm beads, detection decreased to 78-94% for 5 mm collimation/pitch 2 scans when the phantom sections were oriented parallel to the gantry (p = 0.01). Apparent elongation of beads in the slice direction occurred as the collimation and pitch increased. The majority of the elongation (approximately 75%) was attributable to changing the collimator from 3 to 5 mm, with the remainder of the elongation due to doubling the pitch from 1 to 2. CONCLUSION CT scanning at 5 mm collimation and up to pitch 2 is adequate for detection of high contrast lesions as small as 4 mm in this model. However, lesion size and geometry are less accurately depicted than at narrower collimation and lower pitch settings.

Induced pluripotent stem cells: at the heart of cardiovascular precision medicine

The advent of human induced pluripotent stem cell (hiPSC) technology has revitalized the efforts in the past decade to realize more fully the potential of human embryonic stem cells for scientific research. Adding to the possibility of generating an unlimited amount of any cell type of interest, hiPSC technology now enables the derivation of cells with patient-specific phenotypes. Given the introduction and implementation of the large-scale Precision Medicine Initiative, hiPSC technology will undoubtedly have a vital role in the advancement of cardiovascular research and medicine. In this Review, we summarize the progress that has been made in the field of hiPSC technology, with particular emphasis on cardiovascular disease modelling and drug development. The growing roles of hiPSC technology in the practice of precision medicine will also be discussed.