D. Scott Edwards

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.

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.

Comparison of a monomeric and dimeric radiolabeled RGD-peptide for tumor targeting.

The alpha v beta 3 integrin, a transmembrane heterodimeric protein expressed on sprouting endothelial cells, binds to the arginine-glycine-aspartic acid (RGD) amino acid sequence of extracellular matrix proteins such as vitronectin. Growing malignant tumors continuously require angiogenesis. As a result, alpha v beta 3 is preferentially expressed in growing tumors and is a potential target for radiolabeled RGD-peptides. In this study we compared the tumor targeting characteristics of a monomeric radiolabeled RGD-peptide with those of a dimeric analogue. Both peptides were radiolabeled with 99mTc via the hydrazinoni-cotinamid (= HYNIC) moiety to form 99mTc-HYNIC-c(RGDfK) and 99mTc-HYNIC-E-[c(RGDfK)]2. In vitro, the IC50 showed a 10-fold higher affinity of the dimer for the alpha v beta 3 integrin as compared to the monomer (0.1 vs. 1.0 nM). In athymic female BALB/c mice with subcutaneously growing OVCAR-3 ovarian carcinoma xenografts, tumor uptake peaked at 5.8 +/- 0.7% ID/g and 5.2 +/- 0.6% ID/g for the dimer and the monomer, respectively. At 1, 2, and 4 h postinjection (p.i.) uptake of the dimer in the tumor was significantly higher than that of the monomeric analogue. Tumor-to-blood ratios were highest at 24 h p.i. at a value of 63 for both compounds. At all timepoints kidney retention of the dimer was significantly higher as compared to kidney retention of the monomer. In conclusion, in this mouse model the dimeric RGD-peptide showed better retention in the tumor than the monomeric analogue, most likely due to the bivalent interaction with the target cell. Furthermore, kidney retention of the dimeric peptide was higher than that of the monomeric peptide.

Molecular imaging of matrix metalloproteinase in atherosclerotic lesions : resolution with dietary modification and statin therapy

OBJECTIVES This study sought to evaluate the feasibility of noninvasive detection of matrix metalloproteinase (MMP) activity in experimental atherosclerosis using technetium-99m-labeled broad matrix metalloproteinase inhibitor (MPI) and to determine the effect of dietary modification and statin treatment on MMP activity. BACKGROUND The MMP activity in atherosclerotic lesions contributes to the vulnerability of atherosclerotic plaques to rupture. METHODS Atherosclerosis was produced in 34 New Zealand White rabbits by balloon de-endotheliazation of the abdominal aorta and a high-cholesterol diet. In addition, 12 unmanipulated rabbits were used as controls and 3 for blood clearance characteristics. In vivo micro-single-photon emission computed tomography (SPECT) imaging was performed after radiolabeled MPI administration. Subsequently, aortas were explanted to quantitatively measure percent injected dose per gram (%ID/g) MPI uptake. Histological and immunohistochemical characterization was performed and the extent of MMP activity was determined by gel zymography or enzyme-linked immunosorbent assays. RESULTS The MPI uptake in atherosclerotic lesions (n = 18) was clearly visualized by micro-SPECT imaging; MPI uptake was markedly reduced by administration of unlabeled MPI before the radiotracer (n = 4). The MPI uptake was also significantly reduced after diet withdrawal (n = 6) and fluvastatin treatment (n = 6); no uptake was observed in normal control rabbits (n = 12). The %ID/g MPI uptake (0.10 +/- 0.03%) in the atherosclerotic lesions was significantly higher than the uptake in control aorta (0.016 +/- 0.004%, p < 0.0001). Uptake in fluvastatin (0.056 +/- 0.011%, p < 0.0005) and diet withdrawal groups (0.043 +/- 0.011%, p < 0.0001) was lower than the untreated group. The MPI uptake correlated with immunohistochemically verified macrophage infiltration (r = 0.643, p < 0.0001), and MMP-2 (r = 0.542, p < 0.0001) or MMP-9 (r = 0.578, p < 0.0001) expression in plaques. CONCLUSIONS The present data show the feasibility of noninvasive detection of MMP activity in atherosclerotic plaques, and confirm that dietary modification and statin therapy reduce MMP activity.

Molecular Imaging of Activated Matrix Metalloproteinases in Vascular Remodeling

Background— Matrix metalloproteinase (MMP) activation plays a key role in vascular remodeling. RP782 is a novel indium 111In–labeled tracer with specificity for activated MMPs. We hypothesized that RP782 can detect injury-induced vascular remodeling in vivo. Methods and Results— Left common carotid artery injury was induced with a guidewire in apolipoprotein E−/− mice. Sham surgery was performed on the contralateral artery, which served as control for imaging experiments. Carotid wire injury led to significant hyperplasia and expansive remodeling over a period of 4 weeks. MMP activity, detected by in situ zymography, increased in response to injury and was maximal by 3 to 4 weeks after injury. RP782 (11.1 MBq) was injected intravenously into apolipoprotein E−/− mice at 1, 2, 3, and 4 weeks after left carotid injury. MicroSPECT imaging was performed at 2 hours and was followed by CT angiography to localize the carotid arteries. In vivo images revealed focal uptake of RP782 in the injured carotid artery at 2, 3, and 4 weeks. Increased tracer uptake in the injured artery was confirmed by quantitative autoradiography. Pretreatment with 50-fold excess nonlabeled tracer significantly reduced RP782 uptake in injured carotids, thus demonstrating uptake specificity. Weekly changes in the vessel-wall area closely paralleled and correlated with RP782 uptake (Spearman r=0.95, P=0.001). Conclusions— Injury-induced MMP activation in the vessel wall can be detected by RP782 microSPECT/CT imaging in vivo. RP782 uptake tracks the hyperplastic process in vascular remodeling and provides an opportunity to track the remodeling process in vivo.

Molecular Imaging of Matrix Metalloproteinase Expression in Atherosclerotic Plaques of Mice Deficient in Apolipoprotein E or Low-Density-Lipoprotein Receptor

Matrix metalloproteinases (MMPs) are expressed in atherosclerotic plaques and play an important role in plaque instability. Methods: Using 99mTc-labeled broad-spectrum MMP inhibitor (MPI), we performed noninvasive imaging of MMP expression with micro-SPECT/micro-CT in mice deficient in apolipoprotein E (ApoE−/−, n = 14), mice deficient in low-density-lipoprotein receptor (LDLR−/−, n = 14), and C57/BL6 mice as controls (n = 7). Seven ApoE−/− and 7 LDLR−/− received a high-cholesterol diet. After in vivo imaging, aortas were explanted, ex vivo images acquired, and the percent injected dose of MPI per gram (%ID/g) determined, followed by histologic characterization of atherosclerotic lesions. Results: MPI uptake was noninvasively visualized in atherosclerotic lesions by micro-SPECT, with confirmation by micro-CT of anatomic location and aortic calcification. %ID/g in each part of the aorta was highest in ApoE−/− that were fed a high-cholesterol diet, followed by LDLR−/− that were fed a high-cholesterol diet, ApoE−/− that were fed normal chow, and LDLR−/− that were fed normal chow. The control mice had minimal MPI uptake. A significant correlation was noted between %ID/g and % area positive for macrophages (r = 0.81, P = 0.009), MMP-2 (r = 0.65, P = 0.013), and MMP-9 (r = 0.62, P = 0.008). Conclusion: This study demonstrates the usefulness of molecular imaging for noninvasive assessment of the extent of MMP expression in various transgenic mouse models of atherosclerosis receiving a normal or hyperlipidemic diet. It is conceivable that such a strategy may be translationally developed for identification of unstable atherosclerotic plaques.

Molecular Imaging of Matrix Metalloproteinase Activation to Predict Murine Aneurysm Expansion In Vivo

Rupture and dissection are major causes of morbidity and mortality in arterial aneurysm and occur more frequently in rapidly expanding aneurysms. Current imaging modalities provide little information on aneurysm beyond size. Matrix metalloproteinase (MMP) activation plays a key role in the pathogenesis of aneurysm. We investigated whether imaging MMP activation in aneurysm helps predict its propensity to expansion. Methods: We used a model of carotid aneurysm in apolipoprotein E–deficient (apoE−/−) mice. Radiotracers with specificity for activated MMPs were used to detect and quantify MMP activation by micro-SPECT/CT in vivo. Tracer uptake was confirmed by autoradiography and γ-well counting, and specificity was demonstrated using an excess of unlabeled precursor and a specific MMP inhibitor. Results: We demonstrated that several MMPs are expressed with distinct temporal patterns in aneurysm. Significant focal uptake was observed in aneurysmal carotid arteries, peaking at 4 wk after aneurysm induction. In a group of animals imaged serially at 2 and 4 wk after aneurysm induction, MMP tracer uptake at 2 wk correlated well with the vessel area assessed by histology at 4 wk. Conclusion: Molecular imaging of MMP activation is a useful experimental, and potentially clinical, tool to noninvasively predict the propensity of an aneurysm to expansion in vivo.

Fundamentals of Receptor-Based Diagnostic Metalloradiopharmaceuticals

In the last decade, a large number of radiolabeled small biomolecules have been studied for their potential as radiopharmaceuticals for diagnosis and radiotherapy of various diseases. This review will focus on some fundamental aspects of receptor-based diagnostic radiopharmaceuticals, including radiopharmaceutical design, receptors and receptor imaging, choice of biomolecule, and modification of pharmacokinetics.

Effect of an antimicrobial agent on atherosclerotic plaques: assessment of metalloproteinase activity by molecular imaging.

OBJECTIVES Technetium-99m-labeled matrix metalloproteinase inhibitor (MPI) was used for the noninvasive assessment of matrix metalloproteinase (MMP) activity in atherosclerotic plaques after minocycline (MC) intervention. BACKGROUND MMP activity in atherosclerosis contributes to plaque instability. Some antimicrobial agents may attenuate MMP activity. METHODS Atherosclerotic lesions were produced in 38 rabbits with a high cholesterol diet for 4 months; 5 groups of rabbits, in the fourth month, received fluvastatin (FS) (n = 6), low-dose MC (n = 7), high-dose MC (n = 7), a combination of low-dose MC and FS (n = 6), or no intervention (n = 12); 8 unmanipulated rabbits were used as disease controls. Micro-single-photon emission computed tomography imaging was performed in all animals after intravenous MPI administration, followed by pathologic characterization of the aorta. A cell culture study evaluated the effect of MC on MMP production by activated human monocytes. RESULTS MPI uptake was visualized best in untreated atherosclerotic animals (percent injected dose per gram MPI uptake, 0.11 +/- 0.04%). MPI uptake was reduced in the FS (0.06 +/- 0.01%; p < 0.0001), high-dose MC (0.05 +/- 0.01%; p < 0.0001), and MC-FS (0.05 +/- 0.005%; p < 0.0001) groups. Low-dose MC did not resolve MPI uptake significantly (0.08 +/- 0.02; p = 0.167). There was no incremental benefit of the combination of MC and FS. MPI uptake showed a significant correlation with plaque MMP-2, and MMP-9 activity. MMP-9 release from tumor necrosis factor-alpha-activated macrophages was abrogated by incubation with MC. CONCLUSIONS Molecular imaging of MMP activity in atherosclerotic plaque allows for the study of the efficacy of therapeutic interventions. MC administration resulted in substantial reduction in plaque MMP activity and histologically verified plaque stabilization. MC was found to be equally effective as FS.

Matrix Metalloproteinase Activation Predicts Amelioration of Remodeling After Dietary Modification in Injured Arteries

Objective—To establish and validate early noninvasive imaging of matrix metalloproteinase (MMP) activation for monitoring the progression of vascular remodeling and response to dietary modification. Methods and Results—Apolipoprotein E−/− mice that were fed a high-fat diet underwent left common carotid artery wire injury. One week after surgery, a group of animals were withdrawn from the high-fat diet. The other group of animals continued that diet throughout the study. Micro single-photon emission computed tomographic (microSPECT)/CT imaging with RP805 (a 99mTc-labeled tracer targeting activated MMPs) was repeatedly performed at 2 and 4 weeks after surgery. Histological analysis at 4 weeks showed significant left carotid neointima formation, monocyte/macrophage infiltration, and upregulation of several MMPs, which were ameliorated by withdrawal from the high-fat diet. In vivo microSPECT/CT images visualized significant RP805 uptake, reflecting MMP activation, in the injured carotid arteries. MMP activation was reduced as early as 1 week after withdrawal from the high-fat diet and significantly correlated with neointimal area at 4 weeks after surgery. Conclusion—MMP activation predicts the progression of vascular remodeling and can track the effect of dietary modification after vascular injury.