Albert J. Sinusas

Noninvasive imaging of myocardial angiogenesis following experimental myocardial infarction.

Noninvasive imaging strategies will be critical for defining the temporal characteristics of angiogenesis and assessing efficacy of angiogenic therapies. The alphavbeta3 integrin is expressed in angiogenic vessels and represents a potential novel target for imaging myocardial angiogenesis. We demonstrated the localization of an indium-111-labeled ((111)In-labeled) alphavbeta3-targeted agent in the region of injury-induced angiogenesis in a chronic rat model of infarction. The specificity of the targeted alphavbeta3-imaging agent for angiogenesis was established using a nonspecific control agent. The potential of this radiolabeled alphavbeta3-targeted agent for in vivo imaging was then confirmed in a canine model of postinfarction angiogenesis. Serial in vivo dual-isotope single-photon emission-computed tomographic (SPECT) imaging with the (111)In-labeled alphavbeta3-targeted agent demonstrated focal radiotracer uptake in hypoperfused regions where angiogenesis was stimulated. There was a fourfold increase in myocardial radiotracer uptake in the infarct region associated with histological evidence of angiogenesis and increased expression of the alphavbeta3 integrin. Thus, angiogenesis in the heart can be imaged noninvasively with an (111)In-labeled alphavbeta3-targeted agent. The noninvasive evaluation of angiogenesis may have important implications for risk stratification of patients following myocardial infarction. This approach may also have significant clinical utility for noninvasively tracking therapeutic myocardial angiogenesis.

Estimation of 3-D left ventricular deformation from medical images using biomechanical models

The quantitative estimation of regional cardiac deformation from three-dimensional (3-D) image sequences has important clinical implications for the assessment of viability in the heart wall. We present here a generic methodology for estimating soft tissue deformation which integrates image-derived information with biomechanical models, and apply it to the problem of cardiac deformation estimation. The method is image modality independent. The images are segmented interactively and then initial correspondence is established using a shape-tracking approach. A dense motion field is then estimated using a transversely isotropic, linear-elastic model, which accounts for the muscle fiber directions in the left ventricle. The dense motion field is in turn used to calculate the deformation of the heart wall in terms of strain in cardiac specific directions. The strains obtained using this approach in open-chest dogs before and after coronary occlusion, exhibit a high correlation with strains produced in the same animals using implanted markers. Further, they show good agreement with previously published results in the literature. This proposed method provides quantitative regional 3-D estimates of heart deformation.

Low-Flow Ischemia Leads to Translocation of Canine Heart GLUT-4 and GLUT-1 Glucose Transporters to the Sarcolemma In Vivo

BACKGROUND Myocardial ischemia increases heart glucose utilization in vivo. However, whether low-flow ischemia leads to the translocation of glucose transporter (GLUT)-4 and/or GLUT-1 to the sarcolemma in vivo is unknown. METHODS AND RESULTS In a canine model, we evaluated myocardial glucose metabolism in vivo and the distribution of GLUT-4 and GLUT-1 by use of immunoblotting of sarcolemma and intracellular membranes and immunofluorescence localization with confocal microscopy. In vivo glucose extraction increased fivefold (P < .001) and was associated with net lactate release in the ischemic region. Ischemia led to an increase in the sarcolemma content of both GLUT-4 (15 +/- 2% to 30 +/- 3%, P < .02) and GLUT-1 (41 +/- 4% to 58 +/- 3%, P < .03) compared with the nonischemic region and to a parallel decrease in their intracellular contents. Immunofluorescence demonstrated the presence of both GLUT-4 and GLUT-1 on cardiac myocytes. GLUT-1 had a more prominent cell surface pattern than GLUT-4, which was primarily intracellular in the nonischemic region. However, significant GLUT-4 surface labeling was found in the ischemic region. CONCLUSIONS Translocation of the insulin-responsive GLUT-4 transporter from an intracellular storage pool to the sarcolemma occurs in vivo during acute low-flow ischemia. GLUT-1 is also present in an intracellular storage pool from which it undergoes translocation to the sarcolemma in response to ischemia. These results indicate that both GLUT-1 and GLUT-4 are important in ischemia-mediated myocardial glucose uptake in vivo.

On the dark rim artifact in dynamic contrast-enhanced MRI myocardial perfusion studies

A dark band or rim along parts of the subendocardial border of the left ventricle (LV) and the myocardium has been noticed in some dynamic contrast‐enhanced MR perfusion studies. The artifact is thought to be due to susceptibility effects from the gadolinium bolus, motion, or resolution, or a combination of these. Here motionless ex vivo hearts in which the cavity was filled with gadolinium are used to show that dark rim artifacts can be consistent with resolution effects alone. Magn Reson Med, 2005.

Estimation of 3D Left Ventricular Deformation from Echocardiography

The quantitative estimation of regional cardiac deformation from 3D image sequences has important clinical implications for the assessment of viability in the heart wall. Such estimates have so far been obtained almost exclusively from Magnetic Resonance (MR) images, specifically MR tagging. In this paper we describe a methodology for estimating cardiac deformations from 3D echocardiography (3DE). The images are segmented interactively and then initial correspondence is established using a shape-tracking approach. A dense motion field is then estimated using a transversely isotropic linear elastic model, which accounts for the fiber directions in the left ventricle. The dense motion field is in turn used to calculate the deformation of the heart wall in terms of strain in cardiac specific directions. The strains obtained using this approach in open-chest dogs before and after coronary occlusion, show good agreement with previously published results in the literature. They also exhibit a high correlation with strains produced in the same animals using implanted sonomicrometers. This proposed method provides quantitative regional 3D estimates of heart deformation from ultrasound images.

Noninvasive Targeted Imaging of Matrix Metalloproteinase Activation in a Murine Model of Postinfarction Remodeling

Background— Time-dependent activation of matrix metalloproteinases (MMPs) after myocardial infarction (MI) contributes to adverse left ventricular (LV) remodeling; however, noninvasive methods to monitor this process serially are needed. Methods and Results— MMP-targeted radiotracers were developed that displayed selective binding kinetics to the active MMP catalytic domain. Initial nonimaging studies were performed with a 111In-labeled MMP-targeted radiotracer (111In-RP782) and negative control compound (111In-RP788) in control mice (Ctrl) and in mice 1 week after surgically induced MI. Localization of 111In-RP782 was demonstrated within the MI by microautoradiography. A 334±44% increase (P<0.001 versus Ctrl) in relative retention of 111In-RP782 was confirmed by gamma well counting of myocardium. Subsequent high-resolution dual-isotope planar and hybrid micro–single-photon emission computed tomography/CT imaging studies with an analogous 99mTc-labeled MMP-targeted radiotracer (99mTc-RP805) and 201Tl demonstrated favorable biodistribution and clearance kinetics of 99mTc-RP805 for in vivo cardiac imaging, with robust retention 1 to 3 weeks after MI in regions of decreased 201Tl perfusion. Gamma well counting yielded a similar ≈300% increase in relative myocardial retention of 99mTc-RP805 in MI regions (Ctrl, 102±9%; 1 week, 351±77%; 2 weeks, 291±45%; 3 weeks, 292±41%; P<0.05 versus Ctrl). Myocardial uptake in the MI region was also significantly increased ≈5-fold when expressed as percentage injected dose per gram tissue. There was also a significant 2-fold increase in myocardial activity in remote regions relative to control mice, suggesting activation of MMPs in regions remote from the MI. Conclusions— This novel noninvasive targeted MMP radiotracer imaging approach holds significant diagnostic potential for in vivo localization of MMP activation and tracking of MMP-mediated post-MI remodeling.

Multimodality Cardiovascular Molecular Imaging, Part II

Molecular imaging has the potential to profoundly impact preclinical research and future clinical cardiovascular care. In Part I of this 2-part consensus article on multimodality cardiovascular molecular imaging, the imaging methodology, evolving imaging technology, and development of novel targeted molecular probes relevant to the developing field of cardiovascular molecular imaging were reviewed.1 Part II of this consensus article will review the targeted imaging probes available for the identification and evaluation of critical pathophysiological processes in the cardiovascular system. These include novel imaging strategies for the evaluation of inflammation, thrombosis, apoptosis, necrosis, vascular remodeling, and angiogenesis. The current article will also review the role of targeted imaging of a number of cardiovascular diseases, including atherosclerosis, ischemic injury, postinfarction remodeling, and heart failure, as well as the emerging fields of regenerative, genetic, and cell-based therapies. Special emphasis is placed on multimodal imaging, as these hybrid techniques promise to advance the field by combining approaches with complementary strengths and off-setting limitations.2,3 Although some applications of molecular imaging are well established, other clinical applications are under development and still emerging, such as early detection of atherosclerosis or unstable plaque.4 The goals of molecular imaging are to refine risk assessment, facilitate the early diagnosis of disease before the occurrence of debilitating events, aid in the development of personalized therapeutic regimens and to monitor the efficacy of complex therapies. However, to translate the evolving targeted imaging probes, technologies, and applications into clinical care, the imaging community will need to overcome several hurdles. Therefore, the current review will also discuss the opportunities and challenges associated with the implementation and advancement of targeted molecular imaging in clinical practice, and the realization of image-directed personalized medicine.

Small-diameter biodegradable scaffolds for functional vascular tissue engineering in the mouse model.

The development of neotissue in tissue engineered vascular grafts remains poorly understood. Advances in mouse genetic models have been highly informative in the study of vascular biology, but have been inaccessible to vascular tissue engineers due to technical limitations on the use of mouse recipients. To this end, we have developed a method for constructing sub-1mm internal diameter (ID) biodegradable scaffolds utilizing a dual cylinder chamber molding system and a hybrid polyester sealant scaled for use in a mouse model. Scaffolds constructed from either polyglycolic acid or poly-l-lactic acid nonwoven felts demonstrated sufficient porosity, biomechanical profile, and biocompatibility to function as vascular grafts. The scaffolds implanted as either inferior vena cava or aortic interposition grafts in SCID/bg mice demonstrated excellent patency without evidence of thromboembolic complications or aneurysm formation. A foreign body immune response was observed with marked macrophage infiltration and giant cell formation by post-operative week 3. Organized vascular neotissue, consisting of endothelialization, medial generation, and collagen deposition, was evident within the internal lumen of the scaffolds by post-operative week 6. These results present the ability to create sub-1mm ID biodegradable tubular scaffolds that are functional as vascular grafts, and provide an experimental approach for the study of vascular tissue engineering using mouse models.

Noninvasive Imaging of Angiogenesis With a 99mTc-Labeled Peptide Targeted at αvβ3 Integrin After Murine Hindlimb Ischemia

Background—Noninvasive imaging strategies play a critical role in assessment of the efficacy of angiogenesis therapies. The &agr;v&bgr;3 integrin is activated in angiogenic vessels and represents a potential target for noninvasive imaging of angiogenesis. Methods and Results—We evaluated a 99mTc-labeled peptide (NC100692) targeted at &agr;v&bgr;3 integrin for imaging in an established murine model of angiogenesis induced by hindlimb ischemia. Control mice (n=9) or mice with surgical right femoral artery occlusion (n=29) were injected with NC100692 (1.5±0.2 mCi IV) at different times after femoral occlusion (1, 3, 7, and 14 days) for in vivo pinhole planar gamma camera imaging. Tissue from hindlimb proximal and distal to occlusion was excised for gamma well counting and for immunostaining. On in vivo pinhole images, increased focal NC100692 activity was seen distal to the occlusion at days 3 and 7. This increase in relative NC100692 activity was confirmed by gamma well counting. Lectin staining confirmed increased angiogenesis in the ischemic hindlimb at these time points. A fluorescent analogue of NC100692 was used to confirm specificity and localization of the targeted tracer in cultured endothelial cells. In addition, endothelial cell specificity was confirmed on tissue sections with the use of dual immunofluorescent staining of endothelium and the fluorescent analogue targeted at the &agr;v&bgr;3 integrin. Conclusions—A 99mTc-labeled peptide (NC100692) targeted at &agr;v&bgr;3 integrin selectively localized to endothelial cells in regions of increased angiogenesis and could be used for noninvasive serial “hot spot” imaging of angiogenesis. This targeted radiotracer imaging approach is a major advance in tracking therapeutic myocardial angiogenesis and has an important clinical potential.

Detection of Injury-Induced Vascular Remodeling by Targeting Activated αvβ3 Integrin In Vivo

Background—The &agr;vβ3 integrin plays a critical role in cell proliferation and migration. We hypothesized that vascular cell proliferation, a hallmark of injury-induced remodeling, can be tracked by targeting &agr;vβ3 integrin expression in vivo. Methods and Results—RP748, a novel 111In-labeled &agr;vβ3-specific radiotracer, was evaluated for its cell-binding characteristics and ability to track injury-induced vascular proliferation in vivo. Three groups of experiments were performed. In cultured endothelial cells (ECs), TA145, a cy3-labeled homologue of RP748, localized to &agr;vβ3 at focal contacts. Activation of&agr;vβ3 by Mn 2+ led to increased EC binding of TA145. Left common carotid artery wire injury in apolipoprotein E−/− mice led to vascular wall expansion over a period of 4 weeks. RP748 (7.4 MBq) was injected into groups of 9 mice at 1, 3, or 4 weeks after left carotid injury, and carotids were harvested for autoradiography. Relative autographic intensity, defined as counts/pixel of the injured left carotid area divided by counts/pixel of the uninjured right carotid area, was higher at 1 and 3 weeks (1.8±0.1 and 1.9±0.2, respectively) and decreased significantly by 4 weeks after injury (1.4±0.1, P <0.05). Carotid &agr;v and β3 integrin expression was maximal at 1 week and decreased by 4 weeks after injury. The proliferation index, as determined by Ki67 staining, followed a temporal pattern similar to that of RP748 uptake. Dynamic gamma imaging demonstrated rapid renal clearance of RP748. Conclusions—RP748 has preferential binding to activated &agr;vβ3 integrin and can track the injury-induced vascular proliferative process in vivo.