Positron Emission Tomography and Magnetic Resonance Imaging of Cellular Inflammation in Patients with Abdominal Aortic Aneurysms.

OBJECTIVES: Inflammation is critical in the pathogenesis of abdominal aortic aneurysm (AAA) disease. Combined (18)F-fludeoxyglucose ((18)F-FDG) positron emission tomography with computed tomography (PET-CT) and ultrasmall superparamagnetic particles of iron oxide (USPIO)-enhanced magnetic resonance imaging (MRI) are non-invasive methods of assessing tissue inflammation. The aim of this study was to compare these techniques in patients with AAA. MATERIALS AND METHODS: Fifteen patients with asymptomatic AAA with diameter 46 ± 7 mm underwent PET-CT with (18)F-FDG, and T2*-weighted MRI before and 24 hours after administration of USPIO. The PET-CT and MRI data were then co-registered. Standardised uptake values (SUVs) were calculated to measure (18)F-FDG activity, and USPIO uptake was determined using the change in R2*. Comparisons between the techniques were made using a quadrant analysis and a voxel-by-voxel evaluation. RESULTS: When all areas of the aneurysm were evaluated, there was a modest correlation between the SUV on PET-CT and the change in R2* on USPIO-enhanced MRI (n = 70,345 voxels; r = .30; p < .0001). Although regions of increased (18)F-FDG and USPIO uptake co-localised on occasion, this was infrequent (kappa statistic 0.074; 95% CI 0.026-0.122). (18)F-FDG activity was commonly focused in the shoulder region whereas USPIO uptake was more apparent in the main body of the aneurysm. Maximum SUV was lower in patients with mural USPIO uptake. CONCLUSIONS: Both (18)F-FDG PET-CT and USPIO-MRI uptake identify vascular inflammation associated with AAA. Although they demonstrate a modest correlation, there are distinct differences in the pattern and distribution of uptake, suggesting a differential detection of macrophage glycolytic and phagocytic activity respectively.

Investigation of Factors Determining the Enhanced Permeability and Retention Effect in Subcutaneous Xenografts.

Liposomal chemotherapy offers several advantages over conventional therapies, including high intratumoral drug delivery, reduced side effects, prolonged circulation time, and the possibility to dose higher. The efficient delivery of liposomal chemotherapeutics relies, however, on the enhanced permeability and retention (EPR) effect, which refers to the ability of macromolecules to extravasate leaky tumor vessels and accumulate in the tumor tissue. Using a panel of human xenograft tumors, we evaluated the influence of the EPR effect on liposomal distribution in vivo by injection of pegylated liposomes radiolabeled with (111)In. Liposomal accumulation in tumors and organs was followed over time by SPECT/CT imaging. We observed that fast-growing xenografts, which may be less representative of tumor development in patients, showed higher liposomal accumulation than slow-growing xenografts. Additionally, several other parameters known to influence the EPR effect were evaluated, such as blood and lymphatic vessel density, intratumoral hypoxia, and the presence of infiltrating macrophages. The investigation of various parameters showed a few correlations. Although hypoxia, proliferation, and macrophage presence were associated with tumor growth, no hard conclusions or predictions could be made regarding the EPR effect or liposomal uptake. However, liposomal uptake was significantly correlated with tumor growth, with fast-growing tumors showing a higher uptake, although no biological determinants could be elucidated to explain this correlation.

Monitoring Therapy Response of Experimental Arthritis with Radiolabeled Tracers Targeting Fibroblasts, Macrophages, or Integrin αvß3.

UNLABELLED: Rheumatoid arthritis is an autoimmune disease resulting in chronic synovial inflammation. Molecular imaging could be used to monitor therapy response, thus enabling tailored therapy regimens and enhancing therapeutic outcome. Here, we hypothesized that response to etanercept could be monitored by radionuclide imaging in arthritic mice. We tested 3 different targets, namely fibroblast activation protein (FAP), macrophages, and integrin αvß3. METHODS: Male DBA/1J mice with collagen-induced arthritis were treated with etanercept. SPECT/CT scans were acquired at 1, 24, and 48 h after injection of (111)In-RGD2 (integrin αvß3), (111)In-anti-F4/80-A3-1 (antimurine macrophage antibody), or (111)In-28H1 (anti-FAP antibody), respectively, with nonspecific controls included. Mice were dissected after the last scan, and scans were analyzed quantitatively and were correlated with macroscopic scoring. RESULTS: Experimental arthritis was imaged with (111)In-28H1 (anti-FAP), (111)In-anti-F4/80-A3-1, and (111)In-RGD2. Tracer uptake in joints correlated with arthritis score. Treatment decreased joint uptake of tracers from 23 ± 15, 8 ± 4, and 2 ± 1 percentage injected dose per gram (%ID/g) to 11 ± 11 (P < 0.001), 4 ± 4 (P < 0.001), and 1 ± 0.2 %ID/g (P < 0.01) for (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2, respectively. Arthritis-to-blood ratios (in mice with arthritis score 2 per joint) were higher for (111)In-28H1 (5.5 ± 1; excluding values > 25), (111)In-anti-F4/80-A3-1 (10.4 ± 4), and (111)In-RGD2 (7.2 ± 1) than for control (111)In-DP47GS (0.7 ± 0.5; P = 0.002), (111)In-rat IgG2b (0.5 ± 0.2; P = 0.002), or coinjection of excess RGD2 (3.5), indicating specific uptake of all tracers in arthritic joints. CONCLUSION: (111)In-28H1, (111)In-anti-F4/80-A3-1, and (111)In-RGD2 can be used to specifically monitor the response to therapy in experimental arthritis at the molecular level. Further studies, however, still need to be performed.

Subclinical synovitis detected by macrophage PET, but not MRI, is related to short-term flare of clinical disease activity in early RA patients: an exploratory study.

INTRODUCTION: Residual subclinical synovitis can still be present in joints of rheumatoid arthritis (RA) patients despite clinical remission and has been linked to ongoing radiological damage. The aim of the present study was to assess subclinical synovitis by positron emission tomography (PET; macrophage tracer (11)C-(R)-PK11195) in early RA patients with minimal disease activity without clinically apparent synovitis (MDA); and its relationship with clinical outcome and magnetic resonance imaging (MRI), respectively. METHODS: Baseline PET and MRI of hands/wrists were performed in 25 early MDA RA patients (DAS 44 < 1.6; no tender/swollen joints) on combined DMARD therapy. PET tracer uptake (semi-quantitative score: 0-3) and MRI synovitis and bone marrow edema (OMERACT RAMRIS) were assessed in MCP, PIP and wrist joints (22 joints/patient; cumulative score). RESULTS: Eleven of 25 patients (44 %) showed enhanced tracer uptake in ≥ 1 joint. Fourteen of these 25 (56 %) patients developed a flare within 1 year: 8/11 (73 %) with a positive, and 6/14 (43 %) with a negative PET. In the latter, in 5/6 patients flare was located outside the scan region. Median cumulative PET scores of patients with a subsequent flare in the hands or wrists were significantly higher than those of patients without a flare (1.5 [IQR 0.8-5.3] vs 0.0 [IQR 0.0-1.0], p = 0.04); significance was lost when all flares were considered (1.0 [IQR 0.0-4.0] vs 0.0 [IQR 0.0-1.0], p = 0.10). No difference in cumulative MRI scores was observed between both groups. CONCLUSIONS: Positive PET scans were found in almost half of early RA patients with MDA. Patients with a subsequent flare in hand or wrist had higher cumulative PET scores but not MRI scores, suggesting that subclinical arthritis on PET may predict clinical flare in follow-up.

Imaging Macrophage and Hematopoietic Progenitor Proliferation in Atherosclerosis.

RATIONALE: Local plaque macrophage proliferation and monocyte production in hematopoietic organs promote progression of atherosclerosis. Therefore, noninvasive imaging of proliferation could serve as a biomarker and monitor therapeutic intervention. OBJECTIVE: To explore (18)F-FLT positron emission tomography-computed tomography imaging of cell proliferation in atherosclerosis. METHODS AND RESULTS: (18)F-FLT positron emission tomography-computed tomography was performed in mice, rabbits, and humans with atherosclerosis. In apolipoprotein E knock out mice, increased (18)F-FLT signal was observed in atherosclerotic lesions, spleen, and bone marrow (standardized uptake values wild-type versus apolipoprotein E knock out mice, 0.05 ± 0.01 versus 0.17 ± 0.01, P<0.05 in aorta; 0.13 ± 0.01 versus 0.28 ± 0.02, P<0.05 in bone marrow; 0.06 ± 0.01 versus 0.22 ± 0.01, P<0.05 in spleen), corroborated by ex vivo scintillation counting and autoradiography. Flow cytometry confirmed significantly higher proliferation of macrophages in aortic lesions and hematopoietic stem and progenitor cells in the spleen and bone marrow in these mice. In addition, (18)F-FLT plaque signal correlated with the duration of high cholesterol diet (r(2)=0.33, P<0.05). Aortic (18)F-FLT uptake was reduced when cell proliferation was suppressed with fluorouracil in apolipoprotein E knock out mice (P<0.05). In rabbits, inflamed atherosclerotic vasculature with the highest (18)F-fluorodeoxyglucose uptake enriched (18)F-FLT. In patients with atherosclerosis, (18)F-FLT signal significantly increased in the inflamed carotid artery and in the aorta. CONCLUSIONS: (18)F-FLT positron emission tomography imaging may serve as an imaging biomarker for cell proliferation in plaque and hematopoietic activity in individuals with atherosclerosis.

Contrast-enhanced micro-computed tomography using ExiTron nano6000 for assessment of liver injury.

AIM: To explore the potential of contrast-enhanced computed tomography (CECT) using ExiTron nano6000 for assessment of liver lesions in mouse models. METHODS: Three mouse models of liver lesions were used: bile duct ligation (BDL), lipopolysaccharide (LPS)/D-galactosamine (D-GalN), and alcohol. After injection with the contrast agent ExiTron nano6000, the mice were scanned with micro-CT. Liver lesions were evaluated using CECT images, hematoxylin and eosin staining, and serum aminotransferase levels. Macrophage distribution in the injury models was shown by immunohistochemical staining of CD68. The in vitro studies measured the densities of RAW264.7 under different conditions by CECT. RESULTS: In the in vitro studies, CECT provided specific and strong contrast enhancement of liver in mice. CECT could present heterogeneous images and densities of injured livers induced by BDL, LPS/D-GalN, and alcohol. The liver histology and immunochemistry of CD68 demonstrated that both dilated biliary tracts and necrosis in the injured livers could lead to the heterogeneous distribution of macrophages. The in vitro study showed that the RAW264.7 cell masses had higher densities after LPS activation. CONCLUSION: Micro-CT with the contrast agent ExiTron nano6000 is feasible for detecting various liver lesions by emphasizing the heterogeneous textures and densities of CECT images.

Imaging of myocardial inflammation with somatostatin receptor based PET/CT - A comparison to cardiac MRI.

BACKGROUND: Acute myocarditis as well as post-ischemic myocardial inflammation are generally associated with a profound activation of the immune system. Current established imaging techniques such as cardiac MRI reliably demonstrate signs of acute myocardial injury. However, detection of mediating cells such as macrophages is currently limited to experimental settings. We aimed to investigate the feasibility of somatostatin receptor (SSTR) based positron emission tomography/computed tomography (PET/CT) for detecting inflammatory lesions in patients after acute myocardial infarction or acute peri-/myocarditis. METHODS: 12 patients with active peri-/myocarditis (n=6) or sub-acute myocardial infarction (n=6) underwent SSTR-PET/CT and cardiac MRI within 3-10 days after onset of symptoms. The AHA 17-segment model of the left myocardium was used for visual localization of inflamed myocardium for both imaging modalities. Tracer uptake of infarcted/inflamed myocardium was assessed as mean and maximum standardized uptake value (SUVmean and SUVmax) and compared with both remote myocardium and left ventricular (LV) cavity. RESULTS: SSTR-PET/CT revealed areas with increased cardiac tracer uptake in all patients. In the 17-segment model, PET/CT yielded 55 and MRI 47 positive segments. Overall, concordance of the 2 modalities was 85.3% (174/204 segments analyzed). In 9.3% (19/204), more positive segments were identified by PET/CT, whereas in 5.4% (11/204), MRI detected more positive segments. CONCLUSIONS: The imaging patterns of SSTR-directed radiotracers and MRI in vivo show a close spatial relation of macrophage concentration and structural changes. This suggests the possibility of a new potential biomarker that predicts cardiac remodeling and, hence, progression towards heart failure. Prospective trials are warranted.

¹¹¹In-anti-F4/80-A3-1 antibody: a novel tracer to image macrophages.

PURPOSE: Here, the expression of F4/80 on the cell surface of murine macrophages was exploited to develop a novel imaging tracer that could visualize macrophages in vivo. METHODS: The immunoreactive fraction and IC50 of anti-F4/80-A3-1, conjugated with diethylenetriaminepentaacetic acid (DTPA) and radiolabelled with (111)In, were determined in vitro using murine bone marrow-derived macrophages. In vivo biodistribution studies were performed with (111)In-anti-F4/80-A3-1 and isotype-matched control antibody (111)In-rat IgG2b at 24 and 72 h post-injection (p.i.) in SCID/Beige mice bearing orthotopic MDA-MB-231 xenografts. In some studies mice were also treated with liposomal clodronate. Macrophage content in tissues was determined immunohistochemically. Micro-single photon emission computed tomography (SPECT)/CT images were also acquired. RESULTS: In vitro binding assays showed that (111)In-anti-F4/80-A3-1 specifically binds F4/80 receptor-positive macrophages. The immunoreactivity of anti-F4/80-A3-1 was 75 % and IC50 was 0.58 nM. In vivo, injection of 10 or 100 µg (111)In-anti-F4/80-A3-1 resulted in splenic uptake of 78 %ID/g and 31 %ID/g, respectively, and tumour uptake of 1.38 %ID/g and 4.08 %ID/g, respectively (72 h p.i.). Liposomal clodronate treatment reduced splenic uptake of 10 µg (111)In-anti-F4/80-A3-1 from 248 %ID/g to 114 %ID/g and reduced (111)In-anti-F4/80-A3-1 uptake in the liver and femur (24 h p.i.). Tracer retention in the blood and tumour uptake increased (24 h p.i.). Tumour uptake of (111)In-anti-F4/80-A3-1 was visualized by microSPECT/CT. Macrophage density in the spleen and liver decreased in mice treated with liposomal clodronate. Uptake of (111)In-rat IgG2b was lower in the spleen, liver and femur when compared to (111)In-anti-F4/80-A3-1. CONCLUSION: Radiolabelled anti-F4/80-A3-1 antibodies specifically localize in tissues infiltrated by macrophages in mice and can be used to visualize tumours. The liver and spleen act as antigen sink organs for macrophage-specific tracers.

Intrapulmonary administration of bone-marrow derived M1/M2 macrophages to enhance the resolution of LPS-induced lung inflammation: noninvasive monitoring using free-breathing MR and CT imaging protocols.

BACKGROUND: Alveolar macrophages, with their high functional plasticity, were reported to orchestrate the induction and resolution of inflammatory processes in chronic pulmonary diseases. Noninvasive imaging modalities that offer simultaneous monitoring of inflammation progression and tracking of macrophages subpopulations involved in the inflammatory cascade, can provide an ideal and specific diagnostic tool to visualize the action mechanism in its initial stages. Therefore, the purpose of the current study was to evaluate the role of M1 and M2 macrophages in the resolution of lipopolysaccharide (LPS)-induced lung inflammation and monitor this process using noninvasive free-breathing MRI and CT protocols. METHODS: Bone-marrow derived macrophages were first polarized to M1 and M2 macrophages and then labeled with superparamagnetic iron oxide nanoparticles. BALB/c mice with lung inflammation received an intrapulmonary instillation of these ex vivo polarized M1 or M2 macrophages. The biodistribution of macrophages subpopulations and the subsequent resolution of lung inflammation were noninvasively monitored using MRI and micro-CT. Confirmatory immunohistochemistry analyses were performed on lung tissue sections using specific macrophage markers. RESULTS: As expected, large inflammatory areas noninvasively imaged using pulmonary MR and micro-CT were observed within the lungs following LPS challenge. Subsequent intrapulmonary administration of M1 and M2 macrophages resulted in a significant decrease in inflammation starting from 72 h. Confirmatory immunohistochemistry analyses established a progression of lung inflammation with LPS and its subsequent reduction with both macrophages subsets. An enhanced resolution of inflammation was observed with M2 macrophages compared to M1. CONCLUSIONS: The current study demonstrated that ex vivo polarized macrophages decreased LPS-induced lung inflammation. Noninvasive free-breathing MR and CT imaging protocols enabled efficient monitoring of progression and resolution of lung inflammation.

Fluorescence Lifetime Imaging Combined with Conventional Intravascular Ultrasound for Enhanced Assessment of Atherosclerotic Plaques: an Ex Vivo Study in Human Coronary Arteries.

This study evaluates the ability of label-free fluorescence lifetime imaging (FLIm) to complement intravascular ultrasound (IVUS) for concurrent visualization of human coronary vessel composition, structure, and pathology. Co-registered FLIm and IVUS data from 16 coronary segments were correlated to eight distinct pathological features including thin-cap fibroatheroma (TCFA). The sensitivity, specificity, and positive predictive value for combined FLIm-IVUS (89, 99, 89 %) were better than FLIm (70, 98, 88 %) and IVUS (45, 94, 62 %) alone in distinguishing between pathologies. FLIm can assess compositional changes in luminal surface through variations in fluorescence lifetime values (<3.5 ns for lipid-rich areas; >4 ns for collagen-rich areas) enabling detection of macrophages in fibrous caps (sensitivity, 86 %) and distinguishing between relatively stable thick-cap fibroatheromas and rupture-prone TCFA (sensitivity, 80 %) amongst other features. Current results demonstrate the potential of FLIm-IVUS as a new intravascular method for improved evaluation of plaques that may subsequently aid in guiding coronary intervention.

Molecular imaging of inflammation in the ApoE -/- mouse model of atherosclerosis with IodoDPA.

BACKGROUND: Atherosclerosis is a common and serious vascular disease predisposing individuals to myocardial infarction and stroke. Intravascular plaques, the pathologic lesions of atherosclerosis, are largely composed of cholesterol-laden luminal macrophage-rich infiltrates within a fibrous cap. The ability to detect those macrophages non-invasively within the aorta, carotid artery and other vessels would allow physicians to determine plaque burden, aiding management of patients with atherosclerosis. METHODS AND RESULTS: We previously developed a low-molecular-weight imaging agent, [(125)I]iodo-DPA-713 (iodoDPA), which selectively targets macrophages. Here we use it to detect both intravascular macrophages and macrophage infiltrates within the myocardium in the ApoE -/- mouse model of atherosclerosis using single photon emission computed tomography (SPECT). SPECT data were confirmed by echocardiography, near-infrared fluorescence imaging and histology. SPECT images showed focal uptake of radiotracer at the aortic root in all ApoE -/- mice, while the age-matched controls were nearly devoid of radiotracer uptake. Focal radiotracer uptake along the descending aorta and within the myocardium was also observed in affected animals. CONCLUSIONS: IodoDPA is a promising new imaging agent for atherosclerosis, with specificity for the macrophage component of the lesions involved.

Detection of vulnerable atherosclerosis plaques with a dual-modal single-photon-emission computed tomography/magnetic resonance imaging probe targeting apoptotic macrophages.

Atherosclerosis (AS), especially the vulnerable AS plaque rupture-induced acute obstructive vascular disease, is a leading cause of death. Accordingly, there is a need for an effective method to draw accurate predictions about AS progression and plaque vulnerability. Herein we report on an approach to constructing a hybrid nanoparticle system using a single-photon-emission computed tomography (SPECT)/magnetic resonance imaging (MRI) multimodal probe, aiming for a comprehensive evaluation of AS progression by achieving high sensitivity along with high resolution. Ultrasmall superparamagnetic iron oxide (USPIO) was covered by aminated poly(ethylene glycol) (PEG) and carboxylated PEG simultaneously and then functionalized with diethylenetriaminepentacetate acid for (99m)Tc coordination and subsequently Annexin V for targeting apoptotic macrophages abundant in vulnerable plaques. The in vivo accumulations of imaging probe reflected by SPECT and MRI were consistent and accurate in highlighting lesions. Intense radioactive signals detected by SPECT facilitated focus recognization and quantification, while USPIO-based T2-weighted MRI improved the focal localization and volumetry of AS plaques. For subsequent ex vivo planar images, targeting effects were further confirmed by immunohistochemistry, including CD-68 and TUNEL staining; meanwhile, the degree of concentration was proven to be statistically correlated with the Oil Red O staining results. In conclusion, these results indicated that the Annexin V-modified hybrid nanoparticle system specifically targeted the vulnerable AS plaques containing apoptotic macrophages and could be of great value in the invasively accurate detection of vulnerable plaques.

Can the inflammatory response be evaluated using 18F-FDG within zones of microvascular obstruction after myocardial infarction?

UNLABELLED: Inflammation that occurs after acute myocardial infarction plays a pivotal role in healing by facilitating the creation of a supportive scar. (18)F-FDG, which is taken up avidly by macrophages, has been proposed as a marker of cell-based inflammation. However, its reliability as an accurate indicator of inflammation has not been established, particularly in the early postinfarction period when regional myocardial perfusion is often severely compromised. METHODS: Nine adult dogs underwent left anterior descending coronary occlusion with or without reperfusion. Animals were imaged between 7 and 21 d after infarction with PET/MR imaging after bolus injection of gadolinium-diethylenetriaminepentaacetic acid (DTPA), bolus injection of (18)F-FDG, bolus injection of (99)Tc-DTPA to simulate the distribution of gadolinium-DTPA (which represents its partition coefficient in well-perfused tissue), and injection of (111)In-labeled white blood cells 24 h earlier. After sacrifice, myocardial tissue concentrations of (18)F, (111)In, and (99)Tc were determined in a well counter. Linear regression analysis evaluated the relationships between the concentrations of (111)In and (18)F and the dependence of the ratio of (111)In/(18)F to the apparent distribution volume of (99m)Tc-DTPA. RESULTS: In 7 of 9 animals, (111)In increased as (18)F increased with the other 2 animals, showing weak negative slopes. With respect to the dependence of (111)In/(18)F with partition coefficient, 4 animals showed no dependence and 4 showed a weak positive slope, with 1 animal showing a negative slope. Further, in regions of extensive microvascular obstruction, (18)F significantly underestimated the extent of the presence of (111)In. CONCLUSION: In the early post-myocardial infarction period, (18)F-FDG PET imaging after a single bolus administration may underestimate the extent and degree of inflammation within regions of microvascular obstruction.

Biodistribution and PET imaging of a novel [68Ga]-anti-CD163-antibody conjugate in rats with collagen-induced arthritis and in controls.

PURPOSE: The hemoglobin scavenger receptor CD163 is exclusively expressed on monocytes and tissue macrophages, also at sites of inflammation. We examined whether gallium-68 (Ga-68)-labeled anti-CD163-antibody can detect the receptor in vivo. PROCEDURES: We radiolabeled anti-CD163 antibody with Ga-68 and evaluated stability and binding specificity of the conjugate ([(68)Ga]ED2) in vitro. Furthermore, tracer biodistribution was assessed in vivo in healthy rats and rats with acute collagen-induced arthritis (CIA) by MicroPET and tissue analysis. RESULTS: Radiosynthesis of [(68)Ga]ED2 antibody yielded a tracer with high-specific activity and radiochemical purity. [(68)Ga]ED2 bound specifically to CD163 in vitro. In rats, [(68)Ga]ED2 rapidly accumulated in macrophage-rich tissues. The arthritic paws exhibited a low but significant [(68)Ga]ED2 uptake. Interestingly, the systemic distribution was also changed in the sense that a significantly higher liver uptake and lower spleen uptake of [(68)Ga]ED2 was measured in CIA rats that accordingly showed a corresponding change in level of CD163 expression. CONCLUSIONS: [(68)Ga]ED2 specifically binds CD163 in vitro and in vivo. Biodistribution studies in CIA rats suggest that this novel tool may have applications in studies of inflammatory diseases.

PET imaging detection of macrophages with a formyl peptide receptor antagonist.

Macrophages are a major inflammatory cell type involved in the development and progression of many important chronic inflammatory diseases. We previously found that apolipoprotein E-deficient (Apoe(-/-)) mice with the C57BL/6 (B6) background develop type 2 diabetes mellitus (T2DM) and accelerated atherosclerosis when fed a Western diet and that there are increased macrophage infiltrations in pancreatic islets and aorta. The formyl peptide receptor 1 (FPR1) is abundantly expressed on the surface of macrophages. The purpose of this study was to evaluate the applicability of cinnamoyl-F-(D)L-F-(D)L-F (cFLFLF), a natural FPR1 antagonist, to detection of macrophages in the pancreatic islets and aorta. (64)Cu labeled cFLFLF and (18)F-fluorodeoxyglucose (FDG) were administered to mice with or without T2DM. Diabetic mice showed an increased (18)FDG uptake in the subcutaneous fat compared with control mice, but pancreatic uptake was minimal for either group. In contrast, diabetic mice exhibited visually noticeable more cFLFLF-(64)Cu retention in pancreas and liver than control mice. The heart and pancreas isolated from diabetic mice contained more macrophages and showed stronger PET signals than those of control mice. Flow cytometry analysis revealed the presence of macrophages but not neutrophils in pancreatic islets. Real-time PCR analysis revealed much higher FPR1 expression in pancreatic islets of diabetic over control mice. Autoradiography and immunohistochemical analysis confirmed abundant FPR1 expression in atherosclerotic lesions. Thus, (64)Cu-labeled cFLFLF peptide is a more effective PET agent for detecting macrophages compared to FDG.

Glycolaldehyde and maleyl conjugated human serum albumin as potential macrophage-targeting carriers for molecular imaging purposes.

Maleylated bovine serum albumin is a known ligand for targeting macrophages and has potential as a carrier for molecular imaging purposes. We present a novel synthesis of glycolaldehyde-conjugated human serum albumin (GA-HSA) and maleylated human serum albumin (Mal-HSA). Seventeen modifications of fluorescently tagged GA-HSA and Mal-HSA molecules with different degrees of conjugation were prepared. The comparative uptake studies, using 12 of these modifications, were done in vitro on mouse monocytes/macrophages (RAW264.7), and evaluated qualitatively by confocal microscopy and quantitatively by flow cytometry. The GA modifications are taken up by the macrophages approximately 40% better than the maleyl modifications at low concentrations (≤ 3 µM), while at higher concentrations it appears that the maleyl modifications are taken up around 25-44% better than the GA-modified HSA. However, high uptake at low concentrations will be beneficial for in vivo localizing inflammation in areas with low penetration of the probe as in an atherosclerotic plaque. Further, another advantage of GA-HSA is that GA competes less than the maleyl group for the free reactive amine sites that are to be used for conjugation of metal chelating ligands (e.g. tetraazacyclododecanetetraacetic acid and triazacyclononanetriacetic acid). Metal ions such as Gd(3+) and Mn(2+) can be chelated for positive Magnetic Resonance (MR) contrast and positron emitting ions such as (64) Cu(2+) and (68) Ga(3+) for Positron Emission Tomography (PET) imaging. These are important properties, especially, when considering the MR contrast possibilities owing to the low sensitivity of the technique, and would motivate the use of GA-HSA before Mal-HSA.

In vivo monitoring of rat macrophages labeled with poly(l-lysine)-iron oxide nanoparticles.

Coprecipitation of FeCl2 and FeCl3 with aqueous ammonia was used to prepare iron oxide nanoparticles dispersible in aqueous medium. Oxidation of the particles with sodium hypochlorite then yielded maghemite (γ-Fe2 O3 ) nanoparticles which were coated with two types of coating -d-mannose or poly(l-lysine) (PLL) as confirmed by FTIR analysis. The particles were <10 nm according to transmission electron microscopy. Their hydrodynamic particle size was ∼180 nm (by dynamic light scattering). The d-mannose-, PLL-coated, and neat γ-Fe2 O3 particles as well as commercial Resovist® were used to label rat macrophages. The viability and contrast properties of labeled macrophages were compared. PLL-coated γ-Fe2 O3 nanoparticles were found optimal. The labeled macrophages were injected to rats monitored in vivo by magnetic resonance imaging up to 48 h. Transport of macrophages labeled with PLL-γ-Fe2 O3 nanoparticles in rats was confirmed. Tracking of macrophages using the developed particles can be used for monitoring of inflammations and cell migration in cell therapy.

Folate receptor-ß imaging using 99mTc-folate to explore distribution of polarized macrophage populations in human atherosclerotic plaque.

UNLABELLED: In atherosclerotic plaques, the risk of rupture is increased at sites of macrophage accumulation. Activated macrophages express folate receptor-ß (FR-ß), 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 (99m)Tc-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 (99m)Tc-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-ß 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-ß, CD86, and CD163 by flow cytometry. RESULTS: There was a heterogeneous accumulation of (99m)Tc-folate in plaques (median, 2.45 [0.77-6.40] MBq/g). Slices with the highest (99m)Tc-folate accumulation of each plaque showed significantly more expression of FR-ß and CD163, compared with slices with the lowest (99m)Tc-folate accumulation, which showed significantly more expression of iNOS. In in vitro polarized macrophages, messenger RNA expression of FR-ß, 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-ß 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 (99m)Tc-folate accumulation than areas with low accumulation. It is anticipated that (99m)Tc-folate imaging using SPECT as a marker for M2-like macrophages in atherosclerosis might be a good indicator for plaque vulnerability.

Oxidized low-density lipoprotein stimulates macrophage 18F-FDG uptake via hypoxia-inducible factor-1α activation through Nox2-dependent reactive oxygen species generation.

UNLABELLED: For (18)F-FDG PET to be widely used to monitor atherosclerosis progression and therapeutic response, it is crucial to better understand how macrophage glucose metabolism is influenced by the atherosclerotic microenvironment and to elucidate the molecular mechanisms of this response. Oxidized low-density lipoprotein (oxLDL) is a key player in atherosclerotic inflammation that promotes macrophage recruitment, activation, and foam cell formation. We thus explored the effect of oxLDL on macrophage (18)F-FDG uptake and investigated the underlying molecular mechanism including the roles of hypoxia-inducible factor-1α (HIF-1α) and reactive oxygen species (ROS). METHODS: RAW264.7 macrophages were stimulated with native LDL, oxLDL, or lipopolysaccharide. Cells were assessed for (18)F-FDG uptake, lactate production, membrane glucose transporter 1 (GLUT1) expression, and hexokinase activity. ROS generation, Nox expression, and HIF-1α activity were also measured. RESULTS: oxLDL (20 µg/mL) induced a 17.5 ± 1.7-fold increase in macrophage (18)F-FDG uptake by 24 h, which was accompanied by increased lactate production, membrane GLUT1 expression, and hexokinase activity. oxLDL-stimulated (18)F-FDG uptake was completely blocked by inhibitors of Src or phosphoinositide 3-kinase. ROS generation was increased to 262.4% ± 17.9% of controls by oxLDL, and N-acetyl-l-cysteine completely abrogated both oxLDL-induced ROS production and (18)F-FDG uptake. oxLDL increased Nox2 expression, and nicotinamide adenine dinucleotide phosphate oxidase inhibition totally blocked increased ROS generation and (18)F-FDG uptake by oxLDL. Finally, there was a clear ROS-dependent increase of HIF-1α accumulation by oxLDL, and silencing of HIF-1α completely abolished the metabolic effect of oxLDL. CONCLUSION: oxLDL is a strong stimulator of macrophage (18)F-FDG uptake and glycolysis through upregulation of GLUT1 and hexokinase. This metabolic response is mediated by Nox2-dependent ROS generation that promotes HIF-1α activation.

5-Aminolevulinic acid-mediated sonodynamic therapy reverses macrophage and dendritic cell passivity in murine melanoma xenografts.

Sonodynamic therapy (SDT) uses a combination of sonosensitizing drugs and low-intensity therapeutic ultrasound to cause apoptosis and autophagy of tumor cells. However, its effects on the tumor microenvironment, especially on the immune state, remain unknown. In this study, we investigated the transformation of macrophages and dendritic cells (DCs) in the tumor microenvironment during 5-aminolevulinic acid (5-ALA)-mediated SDT in mice transplanted with B16F10 melanomas. Tumor growth and mouse weight were measured. Hematoxylin-eosin staining was used to evaluate tumor morphology to quantify the anti-tumor efficacy of 5-ALA-mediated SDT. We investigated anti-tumor immunity in the tumor microenvironment by immunocytochemical staining of CD68, CD163, CD80, CD86, tumor necrosis factor α (TNF-α), interleukin 10 (IL-10) and interferon γ (IFN-γ). Tumor growth was restrained by 5-ALA-mediated SDT in B16F10 melanoma-bearing mice. CD68 levels increased and CD163 decreased, indicating that M2 macrophages were converted to the M1 phenotype in the tumor. The increase in CD80 and CD86 showed that DCs in the tumor microenvironment tend to mature after SDT treatment. The cytokines INF-γ, TNF-α and IL-10 significantly increased in SDT. Application of low-intensity therapeutic ultrasound alone also led to similar trends in our study, but combined treatment with 5-ALA yielded a change. The original stabilized immune state in the tumor microenvironment can be interrupted by low-intensity therapeutic ultrasound combined with 5-ALA, which enhanced the pro-inflammatory response and reversed the passive properties of macrophages and dendritic cells.