Reza Golestani

Small-animal SPECT and SPECT/CT: application in cardiovascular research

Preclinical cardiovascular research using noninvasive radionuclide and hybrid imaging systems has been extensively developed in recent years. Single photon emission computed tomography (SPECT) is based on the molecular tracer principle and is an established tool in noninvasive imaging. SPECT uses gamma cameras and collimators to form projection data that are used to estimate (dynamic) 3-D tracer distributions in vivo. Recent developments in multipinhole collimation and advanced image reconstruction have led to sub-millimetre and sub-half-millimetre resolution SPECT in rats and mice, respectively. In this article we review applications of microSPECT in cardiovascular research in which information about the function and pathology of the myocardium, vessels and neurons is obtained. We give examples on how diagnostic tracers, new therapeutic interventions, pre- and postcardiovascular event prognosis, and functional and pathophysiological heart conditions can be explored by microSPECT, using small-animal models of cardiovascular disease.

Folate Receptor-β Imaging Using 99mTc-Folate to Explore Distribution of Polarized Macrophage Populations in Human Atherosclerotic Plaque

In atherosclerotic plaques, the risk of rupture is increased at sites of macrophage accumulation. Activated macrophages express folate receptor-β (FR-β), which can be targeted by folate coupled to radioactive ligands to visualize vulnerability. The aim of this study was to explore the presence of activated macrophages in human atherosclerotic plaques by 99mTc-folate imaging and to evaluate whether this technique can discriminate between an M1-like and M2-like macrophage phenotype. Methods: Carotid endarterectomy specimens of 20 patients were incubated with 99mTc-folate, imaged using micro-SPECT, and divided into 3-mm slices. The mean accumulation was calculated per slice, and the distribution of M1-like and M2-like macrophages per slice was quantified by immunohistochemical staining for CD86 as well as inducible nitric oxide synthase (iNOS) for M1 and CD163 and FR-β for M2 macrophages. Monocytes from healthy donors were differentiated toward M1-like or M2-like phenotype by in vitro culturing. Messenger RNA levels of specific M1 and M2 markers were measured by reverse-transcription polymerase chain reaction and expression of FR-β, CD86, and CD163 by flow cytometry. Results: There was a heterogeneous accumulation of 99mTc-folate in plaques (median, 2.45 [0.77–6.40] MBq/g). Slices with the highest 99mTc-folate accumulation of each plaque showed significantly more expression of FR-β and CD163, compared with slices with the lowest 99mTc-folate accumulation, which showed significantly more expression of iNOS. In in vitro polarized macrophages, messenger RNA expression of FR-β, mannose receptor, IL-10, and matrix metalloproteinase-9 was significantly increased in M2-like macrophages, compared with M1-like macrophages. On a receptor level, CD86 was shown to be overexpressed on M1-like macrophages whereas FR-β and CD163 were overexpressed on M2-like macrophages measured by flow cytometry. Conclusion: Higher numbers of M2-like macrophages were present in areas of high 99mTc-folate accumulation than areas with low accumulation. It is anticipated that 99mTc-folate imaging using SPECT as a marker for M2-like macrophages in atherosclerosis might be a good indicator for plaque vulnerability.

Abdominal aortic calcification detected by dual X-ray absorptiometry: A strong predictor for cardiovascular events

Abstract Background. Vertebral fracture assessment (VFA) using dual-energy X-ray absorptiometry can visualize abdominal aortic calcification (AAC). AAC correlates with total atherosclerosis burden. We questioned whether VFA-detected AAC could be used for cardiovascular risk assessment. Methods. VFA images of 2,500 subjects were evaluated to detect and score AAC (n = 164). A random age- and gender-matched set of subjects (n = 325) without AAC served as control group. Patients with prior cardiovascular disease or procedures were excluded. Base-line cardiovascular risk factors and further cardiovascular events were checked. Design-based Cox regression analysis was used to examine the prognostic value of AAC for cardiovascular outcomes. Results. AAC-positive subjects were divided into two groups: low-AAC (score 1–3), and high-AAC group (score > 3). Mean age in the groups was 68, 68, and 71 years, percentage of females was 64.4%, 61%, and 66.1%, and the proportion of cardiovascular events within groups was 1.5%, 6.7%, and 11.9% in control, low-AAC, and high-AAC groups, respectively. Age- and gender-adjusted as well as multivariable analysis showed a significant, higher risk for cardiovascular events incidence in AAC-positive, low-AAC, and high-AAC when compared to the control group. Interpretation. AAC assessed with routine VFA was shown to be a strong predictor for cardiovascular events.

Emergence of molecular imaging of aortic aneurysm: Implications for risk stratification and management

Imaging cellular and molecular processes associated with aneurysm expansion, dissection, and rupture can potentially transform the management of patients with thoracic and abdominal aortic aneurysm. Here, we review recent advances in molecular imaging of aortic aneurysm, focusing on imaging modalities with the greatest potential for clinical translation and application, PET, SPECT, and MRI. Inflammation (e.g., with 18F-FDG, nanoparticles) and matrix remodeling (e.g., with matrix metalloproteinase-targeted tracers) are highlighted as promising targets for molecular imaging of aneurysm. Potential alternative or complementary approaches to molecular imaging for aneurysm risk stratification are briefly discussed.

Feasibility of Vascular Endothelial Growth Factor Imaging in Human Atherosclerotic Plaque Using Zr-89-Bevacizumab Positron Emission Tomography

Intraplaque angiogenesis is associated with the occurrence of atherosclerotic plaque rupture. Cardiovascular molecular imaging can be used for the detection of rupture-prone plaques. Imaging with radiolabeled bevacizumab, a monoclonal anti-vascular endothelial growth factor (VEGF)-A, can depict VEGF levels corresponding to the angiogenic status in tumors. We determined the feasibility of 89Zr-bevacizumab imaging for the detection of VEGF in carotid endarterectomy (CEA) specimens. Five CEA specimens were coincubated with 89Zr-bevacizumab and aspecific 111In-labeled IgG to determine the specificity of bevacizumab accumulation. In 11 CEA specimens, 89Zr-bevacizumab micro-positron emission tomography (PET) was performed following 2 hours of incubation. Specimens were cut in 4 mm wide segments and were stained for VEGF and CD68. In each segment, the mean percent incubation dose per gram of tissue (%Inc/g) and tissue to background ratio were determined. A 10-fold higher accumulation of 89Zr-bevacizumab compared to 111In-IgG uptake was demonstrated by gamma counting. The mean %Inc/ghot spot was 2.2 ± 0.9 with a hot spot to background ratio of 3.6 ± 0.8. There was a significant correlation between the segmental tissue to background uptake ratio and the VEGF score (ρ = .74, p < .001). It is feasible to detect VEGF tissue concentration within CEA specimens using 89Zr-bevacizumab PET. 89Zr-bevacizumab accumulation in plaques is specific and correlates with immunohistochemistry scores.

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.

Adverse cardiovascular effects of anabolic steroids: pathophysiology imaging

Eur J Clin Invest 2012; 42 (7): 795–803

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.

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.