Mahmoud Razavian

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.

Atherosclerosis Plaque Heterogeneity and Response to Therapy Detected by In Vivo Molecular Imaging of Matrix Metalloproteinase Activation

Matrix metalloproteinases (MMPs) play a key role in the development of atherosclerosis and its complications. In vivo detection and quantification of MMP activation can help track the propensity to complications and response to therapy. We sought to establish an in vivo imaging approach for monitoring MMP activation in atherosclerotic mouse aorta and use it to assess the response to dietary modification. Method: Apolipoprotein-deficient mice were fed normal chow or a high-fat diet (HFD) for up to 3 mo or a HFD for 2 mo, followed by 1 mo on normal chow. Then they underwent micro-SPECT/CT, along with autoradiography and oil red O staining of tissues. Results: After 3 mo of HFD, there was considerable atherosclerosis in the aorta. In vivo micro-SPECT/CT using RP782 (an 111In-labeled tracer targeting activated MMPs) showed a heterogeneous pattern of tracer uptake along the aorta. Heterogeneity of RP782 uptake was confirmed by autoradiography, and specificity was demonstrated using excess unlabeled precursor. Tracer uptake quantified by micro-SPECT significantly correlated with uptake quantified by autoradiography. Comparison of oil red O staining with autoradiography demonstrated areas of discordance between plaque presence and tracer uptake. HFD withdrawal led to significant reduction in RP782 uptake beyond the effect on plaque area. MMP expression and macrophage infiltration were similarly heterogeneous along the aorta and significantly reduced after withdrawal from the HFD. Finally, RP782 uptake significantly correlated with aortic macrophage content. Conclusion: Molecular imaging of MMP activation reveals the heterogeneity of atherosclerotic plaques and is a useful tool for tracking plaque biology and response to therapy.

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.

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.

Integrin-Targeted Imaging of Inflammation in Vascular Remodeling

Objective—Inflammation plays a key role in the development of vascular diseases. Monocytes and macrophages express &agr;v&bgr;3 integrin. We used an &agr;v integrin-specific tracer, 99mTc-NC100692, to investigate integrin-targeted imaging for detection vessel wall inflammation. Methods and Results—The binding of a fluorescent homologue of NC100692 to &agr;v&bgr;3 on human monocytes and macrophages was shown by flow cytometry. Vessel wall inflammation and remodeling was induced in murine carotid arteries through adventitial exposure to CaCl2. NC100692 micro single photon computed tomography/CT imaging was performed after 2 and 4 weeks and showed significantly higher uptake of the tracer in CaCl2-exposed left carotids compared with sham-operated contralateral arteries. Histological analysis at 4 weeks demonstrated significant remodeling of left carotid arteries and considerable macrophage infiltration, which was confirmed by real-time polymerase chain reaction. There was no significant difference in normalized &agr;v, &bgr;3, or &bgr;5 mRNA expression between right and left carotid arteries. Finally, NC100692 uptake strongly correlated with macrophage marker expression in carotid arteries. Conclusion—NC100692 imaging can detect vessel wall inflammation in vivo. If further validated, &agr;v-targeted imaging may provide a noninvasive approach for identifying patients who are at high risk for vascular events and tracking the effect of antiinflammatory treatments.

Imaging Vessel Wall Biology to Predict Outcome in Abdominal Aortic Aneurysm

Background—Abdominal aortic aneurysm (AAA) rupture risk is currently determined based on size and symptoms. This approach does not address the rupture risk associated with small aneurysms. Given the role of matrix metalloproteinases (MMPs) in AAA weakening and rupture, we investigated the potential of MMP-targeted imaging for detection of aneurysm biology and prediction of outcome in a mouse model of AAA with spontaneous rupture. Methods and Results—Fifteen-week-old mice (n=66) were infused with angiotensin II for 4 weeks to induce AAA. Saline-infused mice (n=16) served as control. The surviving animals underwent in vivo MMP-targeted micro–single photon emission computed tomographic/computed tomographic imaging, using RP805, a technetium-99m–labeled MMP-specific tracer, followed by ex vivo planar imaging, morphometry, and gene expression analysis. RP805 uptake in suprarenal aorta on micro–single photon emission computed tomographic images was significantly higher in animals with AAA when compared with angiotensin II–infused animals without AAA or control animals. CD68 expression and MMP activity were increased in AAA, and significant correlations were noted between RP805 uptake and CD68 expression or MMP activity but not aortic diameter. A group of angiotensin II–infused animals (n=24) were imaged at 1 week and were followed up for additional 3 weeks. RP805 uptake in suprarenal aorta at 1 week was significantly higher in mice that later developed rupture or AAA. Furthermore, tracer uptake at 1 week correlated with aortic diameter at 4 weeks. Conclusions—MMP-targeted imaging reflects vessel wall inflammation and can predict future aortic expansion or rupture in murine AAA. If confirmed in humans, this may provide a new paradigm for AAA risk stratification.

Multimodality and Molecular Imaging of Matrix Metalloproteinase Activation in Calcific Aortic Valve Disease

Calcific aortic valve disease (CAVD) is the most common cause of aortic stenosis. Matrix metalloproteinases (MMPs) are upregulated in CAVD and contribute to valvular remodeling and calcification. We investigated the feasibility and correlates of MMP-targeted molecular imaging for detection of valvular biology in CAVD. Methods: Apolipoprotein E–deficient (apoE−/−) mice were fed a Western diet (WD) for 3, 6, and 9 mo (n = 108) to induce CAVD. Wild-type mice served as the control group (n = 24). The development of CAVD was tracked with CT, echocardiography, MMP-targeted small-animal SPECT imaging using 99mTc-RP805, and histologic analysis. Results: Key features of CAVD—leaflet thickening and valvular calcification—were noted after 6 mo of WD and were more pronounced after 9 mo. These findings were associated with a significant reduction in aortic valve leaflet separation and a significant increase in transaortic valve flow velocity. On in vivo SPECT/CT images, MMP signal in the aortic valve area was significantly higher at 6 mo in WD mice than in control mice and decreased thereafter. The specificity of the signal was demonstrated by blocking, using an excess of nonlabeled precursor. Similar to MMP signal, MMP activity as determined by in situ zymography and valvular inflammation by CD68 staining were maximal at 6 mo. In vivo 99mTc-RP805 uptake correlated significantly with MMP activity (R2 = 0.94, P < 0.05) and CD68 expression (R2 = 0.98, P < 0.01) in CAVD. Conclusion: MMP-targeted imaging detected valvular inflammation and remodeling in a murine model of CAVD. If this ability is confirmed in humans, the technique may provide a tool for tracking the effect of emerging medical therapeutic interventions and for predicting outcome in CAVD.

Molecular Imaging of Vascular Endothelial Growth Factor Receptors in Graft Arteriosclerosis

Objective—Vascular endothelial growth factor (VEGF) signaling plays a key role in the pathogenesis of vascular remodeling, including graft arteriosclerosis. Graft arteriosclerosis is the major cause of late organ failure in cardiac transplantation. We used molecular near-infrared fluorescent imaging with an engineered Cy5.5-labeled single-chain VEGF tracer (scVEGF/Cy) to detect VEGF receptors and vascular remodeling in human coronary artery grafts by molecular imaging. Methods and Results—VEGF receptor specificity of probe uptake was shown by flow cytometry in endothelial cells. In severe combined immunodeficiency mice, transplantation of human coronary artery segments into the aorta followed by adoptive transfer of allogeneic human peripheral blood mononuclear cells led to significant neointima formation in the grafts over a period of 4 weeks. Near-infrared fluorescent imaging of transplant recipients at 4 weeks demonstrated focal uptake of scVEGF/Cy in remodeling artery grafts. Uptake specificity was demonstrated using an inactive homolog of scVEGF/Cy. scVEGF/Cy uptake predominantly localized in the neointima of remodeling coronary arteries and correlated with VEGF receptor-1 but not VEGF receptor-2 expression. There was a significant correlation between scVEGF/Cy uptake and transplanted artery neointima area. Conclusion—Molecular imaging of VEGF receptors may provide a noninvasive tool for detection of graft arteriosclerosis in solid organ transplantation.

Lipid lowering and imaging protease activation in atherosclerosis

BackgroundLipid lowering is a mainstay of modern therapeutic approach to atherosclerosis. We sought to evaluate matrix metalloproteinase (MMP)-targeted microSPECT imaging for tracking of the effect of lipid-lowering interventions on plaque biology in atherosclerotic mice in vivo.Methods and ResultsApoE−/− mice fed on a high fat diet (HFD) for 2 months were randomly assigned to continuation of HFD, HFD plus simvastatin, HFD plus fenofibrate and high fat withdrawal (HFW). The animals underwent serial microSPECT/CT imaging using RP805, a 99mTc-labeled MMP-targeted tracer at 1 and 4 weeks after randomization. All three interventions reduced total blood cholesterol by 4 weeks. In animals on HFD, aortic arch RP805 uptake significantly increased from 1 week to 4 weeks. Tracer uptake in fenofibrate and HFW groups was significantly lower than uptake in the HFD group at 4 weeks. Similarly, CD 68 gene expression, reflecting plaque inflammation, was significantly lower in fenofibrate and HFW groups compared to HFD group. MMP tracer uptake significantly correlated with aortic CD68, but not VE-cadherin or smooth muscle α-actin expression.ConclusionsMMP tracer uptake paralleled the effect of lipid-lowering interventions on plaque inflammation in atherosclerotic mice. MMP-targeted imaging may be used to track the effect of therapeutic interventions in atherosclerosis.

Matrix Metalloproteinase–Targeted Imaging of Lung Inflammation and Remodeling

Imaging techniques for detection of molecular and cellular processes that precede or accompany lung diseases are needed. Matrix metalloproteinases (MMPs) play key roles in the development of pulmonary pathology. The objective of this study was to investigate the feasibility of in vivo MMP-targeted molecular imaging for detection of lung inflammation and remodeling. Methods: Lung-specific IL-13 transgenic (Club cell 10-kDa protein [CC10]-IL-13 Tg) mice and wild-type littermates were used in this study. Lung structure, gene expression, and MMP activity were assessed by histology, real-time reverse transcription polymerase chain reaction, Western blotting, and zymography. MMP activation was imaged by in vivo small-animal SPECT/CT followed by ex vivo planar imaging. Signal specificity was addressed using a control tracer. The correlation between in vivo MMP signal and gene expression was addressed. Results: CC10-IL-13 Tg mice developed considerable pulmonary tissue remodeling and inflammation. CD68, MMP-12, and MMP-13 were significantly higher in CC10-IL-13 Tg lungs. On in vivo small-animal SPECT/CT and ex vivo planar images, the MMP signal was significantly higher in the lungs of CC10-IL-13 Tg mice than wild-type animals. Furthermore, a nonbinding analog tracer showed significantly lower accumulation in CC10-IL-13 Tg lungs relative to the specific tracer. There was a significant correlation between small-animal SPECT/CT–derived MMP signal and CD68 expression in the lungs (r = 0.70, P < 0.01). Conclusion: Small-animal SPECT/CT–based MMP-targeted imaging of the lungs is feasible and reflects pulmonary inflammation. If validated in humans, molecular imaging of inflammation and remodeling can potentially help early diagnosis and monitoring of the effects of therapeutic interventions in pulmonary diseases.