Dual molecular imaging for targeting metalloproteinase activity and apoptosis in atherosclerosis: molecular imaging facilitates understanding of pathogenesis
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Macrophage apoptosis and MMP activity contribute to vulnerability of atherosclerotic plaques to rupture. By employing molecular imaging techniques, we investigated if apoptosis and MMP release are interlinked.
Atherosclerosis was produced in rabbits receiving high-cholesterol diet (HC), who underwent dual radionuclide imaging with 99mTc-labeled matrix metalloproteinase inhibitor (MPI) and 111In-labeled annexin A5 (AA5) using micro-SPECT/CT. %ID/g MPI and AA5 uptake was measured, followed by histological characterization. Unmanipulated animals were used as disease controls. Correlation between MPI and AA5 uptake was undertaken and relationship confirmed in culture study of activated THP-1 monocytes.
MPI and AA5 uptake was best visualized in HC diet animals (n = 6) and reduced significantly after fluvastatin treatment (n = 4) or diet withdrawal (n = 3). %ID/g MPI (.087 ± .018%) and AA5 (.03 ± .01%) uptake was higher in HC than control (n = 6) animals (.014 ± .004%, P < .0001; .0007 ± .0002%, P < .0001), and reduced substantially after diet or statin intervention. There was a significant correlation between MPI and AA5 uptake (r = .62, P < .0001), both correlated with pathologically verified MMP-9 activity, macrophage content, and TUNEL staining. In vitro studies demonstrated MMP-9 release in culture medium from apoptotic THP-1 monocytes.
The present study suggests that apoptosis and MMP are interrelated in atherosclerotic lesions and the targeting of more than one molecular candidate is feasible by molecular imaging.
KeywordsRadionuclides SPECT vulnerable atherosclerotic plaque
In two-thirds of patients presenting with acute coronary syndromes, coronary thrombosis is associated with rupture of the fibrous cap of an atherosclerotic plaque.1 The triggers for the rupture include prevalence of monocyte-macrophage infiltration and their apoptosis as well as cytokine production including matrix metalloproteinases (MMP). Almost one half of the macrophages at the site of plaque rupture demonstrate evidence of apoptosis with activation of caspase-1.2 In vitro experiments have confirmed the role of caspase-1 in induction of apoptosis and the use of caspase-1 inhibitor (YVAD) substantially reduced the apoptosis of macrophages in an experimental model of atherosclerosis.3 On the other hand, macrophage infiltration in the plaque is associated with MMP expression and in turn to degradation of extracellular matrix and cap rupture.4
In addition to acting as a trigger to cap rupture, macrophage apoptosis and MMP activity contribute to plaque vulnerability. Apoptosis of macrophages in necrotic core5 leads to enlargement of the core size; greater the necrotic core volume more prone is the plaque to rupture.6 On the other hand, the increase in MMP in the necrotic core promotes expansive outward remodeling of the plaque,7 and renders the plaque susceptible to rupture. These two processes, which are independently critical for plaque rupture, are both related to macrophage infiltration and it is logical to presume that these processes are interrelated. The apoptosis of cellular components in an atherosclerotic plaque is no longer considered an inert process. Fas-induced apoptosis of smooth muscle cell (SMC) has been shown to produce cytokines such as monocyte-chemoattractant protein 1 (MCP-1), CINC/IL-8, and other pro-inflammatory genes resulting in an extensive macrophage infiltration.8 Similar to the SMC-related release of cytokines, it is possible that apoptosis of macrophages may also be associated with secretory activity.
To evaluate if apoptosis of macrophages is linked to MMP and to determine the correlation of macrophage apoptosis and MMP release in vivo, we performed noninvasive imaging with 111In-annexin A5 (AA5; as a marker of apoptosis) and 99mTc-labeled broad-spectrum matrix metalloproteinase inhibitor (MPI; as a marker of MMP activity) in rabbits with experimentally induced atherosclerotic lesions. We also evaluated the interrelation by inducing apoptosis of activated monocytes and investigating whether the MMP activity was increased in the culture medium.
Noninvasive imaging was performed in atherosclerotic rabbits for the detection of apoptosis process and MMP activity by employing 111In-AA5 (which targets phosphatidyl serine expression on the apoptotic cells) and 99mTc-MPI (which binds to activated catalytic domain of MMP). The correlation of the two processes was confirmed in the in vitro experiments using PMA-activated human monocytic leukemic cell line, THP-1. The THP-1 cells were transfected with caspase-1 gene, and MMP production was determined in the culture medium. MMP-9 production and activity were measured by gel zymography as reported previously by ELISA.9 The effects of caspase-1 inhibitor and endoplasmatic reticulum (ER) stress inducers were also evaluated.
Atherosclerosis was produced experimentally in rabbits by aortic deendothelialization and high-cholesterol (HC) diet. To obtain a large range of macrophage infiltration, apoptosis, and MMP production in the atherosclerotic lesions, the hypercholesterolemic rabbits were subjected to dietary modification or treated with fluvastatin. This protocol conforms to the Guidelines for the Care and Use of Laboratory Animals by the US National Institute of Health (NIH Publication No. 85-23, revised 1996) and has been approved by the Institutional Laboratory Animal Care and Use Committee at University of California, Irvine.
In Vivo Experimental Design
The in vivo study was performed in 19 New Zealand white (NZW) rabbits. Of the 19 rabbits, 13 rabbits were subjected to balloon deendothelialization of their abdominal aorta and were fed HC diet for 4 months to induce atherosclerotic lesions. Of these, 3 atherosclerotic rabbits were returned to normal chow in the last fourth month and 4 received fluvastatin (1 mg/kg p.o., once a day) in the last month. Dual imaging was performed after simultaneous intravenous administration of Tc-99m-labeled MPI (257.2 ± 3.7 MBq/6.95 ± 0.10 mCi) and In-111-labeled annexin A5 (17.0 ± 6.29 MBq/0.46 ± 0.17 mCi). The remaining 6 rabbits were left unmanipulated for 4 months and fed normal chow. These animals were used for dual imaging as disease controls.
Induction of atherosclerosis
Male NZW rabbits (2.5-3.5 kg) obtained from Western Oregon Breeding Laboratories (Philomoth, OR) were started on 0.5% cholesterol and 6% peanut oil diet. One week later, balloon deendothelialization of the abdominal aorta was performed with a 4 F Fogarty embolectomy catheter (12-040-4F; Edwards Lifesciences LLC, Irvine, CA) as described previously.10 For this purpose, animals were anesthetized with a mixture of ketamine and xylazine. The right femoral artery was surgically exposed, an embolectomy catheter was introduced through the femoral arteriotomy and advanced retrograde in the aorta up to the level of the diaphragm. The catheter was inflated and endothelial denudation of the abdominal aorta was performed by pulling down the inflated catheter to the bifurcation of the aorta. Three such passes were made. The femoral artery was then ligated, and the incision site closed. The high-cholesterol, high-fat diet was continued for 15 more weeks.
Imaging agents and radiolabeling
Two targeting agents radiolabeled with two different isotopes were employed for imaging. First, MPI (RP-805, kind gift of Lantheus Imaging Inc., North Billerica, MA) radiolabeled with technetium-99m was used. The chemical structure of MPI has been reported previously.9,11 MPI binds specifically to the activated catalytic domain of a broad range of MMP and not other proteases,12 and therefore has high enzyme inhibitory profile.9 For radiolabeling of MPI, the contents of the vehicle vial were dissolved in 0.5 mL of 0.9% sterile saline. The clear solution was transferred to a vial containing 25-35 μg of MPI. Subsequently the mixture was incubated for 10 minutes to dissolve all particles and ~3.33 GBq (90 mCi/mL) of 99mTc pertechnetate was added. The reaction vial was heated at 100°C for 10 minutes. Then HPLC analysis was performed to check the radioefficiency, which revealed product peak of >97%.
Second, AA5 binds to phosphatidylserin expressed on the surface of apoptotic cells with a nanomolar affinity. Normally, AA5 has been radiolabeled with Tc-99m and employed successfully for experimental and clinical imaging of apoptosis noninvasively. For this study, we developed a novel imaging strategy by labeling AA5 with indium-111. For radiolabeling AA5, 55.5 MBq (1.5 mCi) of 111In was added to 30 μg of AA5-DTPA conjugate (kindly provided by PharmaTarget Inc, Maastricht, Netherlands). After 30 minutes of incubation, instant thin layer chromatography was performed.
In vivo and ex vivo imaging protocols
Radionuclide imaging was performed immediately and 4 hours after administration of radiotracers using a dual-head micro-SPECT gamma camera combined with micro-CT (X-SPECT, Gamma Medica, Inc., Northridge, CA). SPECT images of the aorta were acquired in a 64 × 64 matrix, 32 stops at 20 seconds (at 0 hour) or 120 seconds (at 4 hours) per stop first at 140 keV photopeak of 99mTc with 15% windows using a low-energy, high-resolution parallel-hole collimator and second at 247 keV photopeak of 111In with 15% windows using a medium energy parallel-hole collimator. Immediately after SPECT imaging, a micro-CT scan was acquired using an X-ray tube operating at 50 kVp and 0.6 mA. Images were captured for 2.5 seconds per view for 256 views in 360° rotation. After transferring to 256 × 256 matrix, the micro-SPECT images and micro-CT tomographic studies were fused. Animals were killed after in vivo imaging, with an overdose of sodium pentobarbital (120 mg/kg). Ex vivo gamma imaging of the excised aortas was performed. The explanted aortas were imaged for 30 minutes. After ex vivo imaging, the aortas were segmented at 1-cm intervals, weighed, and gamma counted in an automatic well-type gamma counter (Perkin Elmer Wallac Inc., Gaithersburg, MD) for calculation of the percent total injected dose per gram tissue (%ID/g) uptake. Aortas were then preserved for histologic and immunohistochemical investigation.
Tissue samples of the main organs were used for calculation of the %ID/g uptake to evaluate the biodistribution. To correct for the radioactive decay and permit calculation of the concentration of radioactivity as a fraction of the administered dose, aliquots of the injected dose were counted simultaneously.
Histological and immunohistochemical evaluation
One-half of every 1 cm aortic segment was snap frozen and the other half was fixed overnight with 4% paraformaldehyde in PBS, pH 7.4 at 4°C. The first half of the each specimen was subdivided into three equidistant sections, dehydrated in a graded series of ethanol, and embedded in paraffin for further processing in triplicate. Paraffin blocks were cut in 4 μm thick sections, floated on a water bath containing deionized water (43°C), and mounted on vectabond reagent-treated slides (Vectabond, SP-1800, Vector Labs, Burlingame, CA). After deparaffinization by heating (25 minutes at 56°C) and dehydration using xylene and graded series of ethanol, the tissue sections were stained with hematoxylin & eosin and Movat pentachrome stains. Histologic specimens were analyzed using a classification scheme based on the recommendations of the American Heart Association (AHA).13
As described previously,9,14 immunohistochemistry was performed by standard staining procedures. Smooth muscle cell (SMC) was identified using a primary antibody against α-actin isotypes (MAB1420, 1:10,000, R&D Systems, Minneapolis, MI) and macrophages by RAM-11 (M 0633, 1:3,000, DAKO, Carpinteria, CA). For MMP-9 staining in atherosclerotic lesions monoclonal MMP-9 (gelatinase-B, IM37, 1:15,000, Calbiochem) antibody was used. Color reaction was developed by Novared substrate-chromogen system (SK-4800, red color, Vector Lab) and diaminobenzidine kit (SK-4100, DAB, brown color, Vector Lab, Burlingame, CA). The sections were counterstained with Gill’s hematoxylin. Sections incubated in parallel without primary antibody or control IgG were used as negative control. Immunostaining was observed (Zeiss Axiovert-200 microscope, Carl Zeiss, Thornwood, NY) and images captured (Zeiss Axiocam high-resolution digital color camera, 1,300 × 1,030 pixels using Axiovision 3.1 software, Thornwood, NY). Digital images were analyzed using Image-Pro Plus version 5.0 (Media Cybernetics, Bethesda, MD). The percentage of immunopositive area (immunopositive area/total intimal area × 100) was calculated.
In situ labeling of DNA fragmentation was performed using terminal deoxyribonucleotide transferase (TdT)-mediated nick-end labeling based on an in situ apoptosis detection kit (TACS, Trevigen). Deparaffinized sections were treated with 0.3% hydrogen peroxide for 10 minutes for inactivation of endogenous peroxidase. The sections were rinsed and then digested with 20 μg of proteinase K (EM Science). Exposed DNA fragments were labeled with biotinylated nucleotides (dNTPs) and TDT for 1 hour at 37°C. The incorporation of biotinylated nucleotides into DNA was detected with a streptavidin-conjugated horseradish peroxidase. A positive reaction was visualized with the chromogenic substance diaminobenzidine tinted with CoCl2, producing a black reaction product. The sections were counterstained with methylgreen (blue-green nuclei). The number of positive cells was counted in the whole intima of each section.
In Vitro Experimental Design
Cell culture studies
The in vitro experiments were performed with human monocytic leukemic cell line (THP-1). Cells were grown in RPMI medium 1640 supplemented with 10% heat-inactivated fetal bovine serum, 100 units/mL penicillin, 100 μg/mL streptomycin, and 2 mM l-glutamine at 37°C in 5% CO2.
Recombinant plasmid vectors
A full-length cDNA of CASP1 was isolated from ICE-α pBluescript KS plasmid15 and was cloned into the Xba1-EcoR1 site of eukaryotic expression vector pCDNA3 (Invitrogen corporation, Carlsbad, CA). Authenticity of the clone was confirmed by DNA sequencing. Empty control vector pcDNA3 and cDNA of CASP1 cloned in reverse orientation into the BamH1-EcoR1 site of pcDNA3 were used as negative controls.
Electroporation of THP-1 cells
Exponentially growing THP-1 cells (1 × 107 cells) were washed and resuspended in 300 μL complete media containing 20 mM Hepes. Cell suspension was mixed with 10 μg of pCDNA3-CASP1, pCDNA3-CASP1-as, or pCDNA3 plasmid DNA and transferred to a cuvette (0.4-cm gap; BioRad Laboratories) and electroporated (230 V, 960 μF, Gene Pulsar II, BioRad Laboratories, Hercules, CA). Electroporated cells were diluted with 10 mL of RPMI 1640 containing 10% fetal bovine serum, 100 IU/mL of penicillin, 100 μg/mL of streptomycin, and 0.6 mg/mL of glutamine, and grown at 37°C for 48 hours. MMP-2 and -9 productions were estimated in the culture medium by gel zymography.16 Release of MMP-9 and IL1-β in the culture medium was also measured by activity assay and ELISA. The effect of a caspase-1 inhibitor and an ER stress inducer on MMP production was also evaluated.
Culture supernatants were mixed with SDS-sample buffer (50 mM Tris-HCl pH 6.8, 10% glycerol, 1% SDS, 0.01% bromophenol blue) in the absence of reducing agent and electrophoresed in a 7.5% SDS-polyacrylamide gel containing 0.1% (w/v) gelatin. The gel was incubated at room temperature for 1 hour in 2.5% TritonX 100 and subsequently at 37°C overnight in a buffer containing [10 mM CaCl2, 150 mM NaCl, and 50 mM Tris (pH 7.5) 0.02% NaN3]. The gel was then stained for protein with 0.25% Coomassie. Gelatinolytic activity was detected as a clear zone in a dark field.
Matrix metalloproteinase activity assays
For MMP assay, MMP-9 Biotrak Activity Assay System and Interleukin-1 β Biotrak ELISA system (GE Healthcare Bio-Sciences, Piscataway, NJ) were used according to manufacturer’s protocol. THP-1 culture medium was collected for MMP assay after 48 hours of transfection or 24-48 hours treatment with different inducers.
Cell-permeable caspase-1 inhibitor YVAD-CHO, caspase-3 inhibitor DEVD-CHO, and broad-spectrum caspase inhibitor Z-VAD-FMK, (BIOMOL International, Plymouth Meeting, PA) were used. 7-ketocholesterol, cholesterol-5β, ionomycin, and thapsigargin were purchased from Sigma Chemical Co. (St. Louis, MO).
All results are presented as the mean ± SD. To determine the statistical significance of differences between groups one-way ANOVA was used followed by post hoc analysis using Fisher’s PLSD. P value of <.05 was considered statistically significant. Blood radioactivity of MPI and AA5 was presented as the mean (±SD) percent ID/g of tissue and was plotted as a function of time after injection. The clearance curves were fitted to biexponential functions by nonlinear least squares.
Noninvasive Detection of MMP Expression and Apoptosis in Atherosclerotic Lesions
Quantitative MPI and AA5 Uptake
The percent ID/g uptake of MPI in the atherosclerotic lesions of the animals on uninterrupted high-cholesterol diet (0.087 ± 0.018%) was significantly higher than the uptake in the abdominal aorta of disease control animals (0.014 ± 0.004%; P < .0001). The quantitative MPI uptake in diet-withdrawal group (0.047 ± 0.005%; P < .0005) and fluvastatin-treated animals (0.053 ± 0.013%; P < .01) was significantly lower compared to uninterrupted HC diet group (0.087 ± 0.018%) (Figure 2B). Similarly, the percent ID/g AA5 uptake in the atherosclerotic lesions of animals with uninterrupted HC diet (0.03 ± 0.01%) was significantly higher than the uptake in disease control rabbits (0.0007 ± 0.0002%; P < .0001). The quantitative uptake of AA5 in diet-withdrawal group (0.018 ± 0.004%; P < .05) and fluvastatin-treated groups (0.018 ± 0.01%; P < .05) was significantly lower compared to uninterrupted HC diet group (Figure 2B).
There was a strong correlation between MPI and AA5 uptake in the atherosclerotic lesions (r = 0.62, P < .0001) (Figure 2C).
Histopathologic Characterization of Atherosclerotic Lesions
In Vitro Culture Studies
We also evaluated the role of oxysterol, cholesterol-5β, and 7-ketocholesterol, present in atherosclerotic lesions and reported to induce SMC death and plaque destabilization, on MMP-9. THP-1 cells treated with 40 μg/mL of 7-ketocholesterol and 30 μM of cholesterol-5β induced the expression and release of MMP-9 (Figure 5D). Apoptosis induced by oxLDL is mediated through Ca2+ influx and Ca2+ deregulation. THP-1 cells treated with 1 μM thapsigargin, an inhibitor of sarcoplasmic reticulum Ca2+ ATPase (SERCA) which raises cytosolic calcium concentration also induced higher MMP-9 release (Figure 5D). But ionomycin, a potent and selective Ca2+ ionophore, which acts primarily at the level of the internal Ca2+ stores and enhances Ca2+ influx, had no effect on MMP-9 expression level (Figure 5D). Lipopolysaccharide (LPS) treatment induced higher MMP-9 expression and extracellular release into the medium, but exactly similar to CASP1 overexpression had no effect on MMP-2 level (Figure 5E). MMP-2 and -9 levels remained unchanged in interferon-gamma (IFN-γ)-treated THP-1 cells.
The direct relationship between macrophage apoptosis and MMP release was observed in imaging experiments, which was maintained over the broad range of prevalence of apoptosis and MMP production. Both, the MMP production and apoptosis were substantially reduced in diet-withdrawal and statin treatment groups. MPI and AA5 uptake correlated with pathologically verified apoptosis and macrophage, and MMP-9 positive areas in the atherosclerotic lesions. This relationship was confirmed in culture experiments. Both, in vitro and in vivo experiments demonstrated that macrophage apoptosis and MMP production may be intimately related and highlight the importance of inflammation in plaque vulnerability.
Macrophage Apoptosis and MMP Release
Although not evaluated in atherosclerosis, neurons under cellular stress undergo apoptosis and release MMP-3.17 The MMP-3 upregulation occurs upstream of caspase-3 (CASP3) activation but downstream of c-Jun N-terminal protein kinase (JNK) in the apoptosis signaling cascade. C-Jun, phosphorylated by JNK, acts as a transcription factor for MMP-3 gene. However, our experiments suggest that apoptosis may be upstream to MMP-9 release. Differentiated versus undifferentiated cell types, CASP3 versus CASP1, and MMP-3 versus MMP-9 may explain some of the differences. Our study suggests that CASP1, besides its pro-inflammatory actions, has pro-apoptotic potential.3 In patients dying of acute coronary events, extensive apoptosis of macrophages, CASP1 activation, and increased MMP-9 activity have been demonstrated at the site of plaque rupture.2 We have also observed CASP1-mediated apoptosis of macrophages in advanced experimental atherosclerosis in a rabbit model.3 On the other hand, a possible role of MMP in inducing apoptosis in atherosclerosis is unclear. Besides extracellular matrix proteins, MMP are known to degrade integrins,18 and may promote apoptosis of SMC by cell-surface N-cadherin shedding. Also by liberating nonmatrix substrates such as growth factors from attachment to matrix components or cell surface, MMP can influence apoptosis.19 MMP can modulate apoptotic signaling by release of FasL20 and TNF-α21 from cell surface.
Active MMP-3 has also been found in the nucleus of several cultured cell types and may be associated with an increased rate of apoptosis.22 Pro-MMP-2 has been found in the nucleus of cardiomyocytes; activated nuclear MMP-2 results in DNA repair enzyme poly-ADP ribose-polymerase (PARP) fragmentation and may increase genetic instability to trigger apoptosis.23 In adrenergic receptor-stimulated rat cardiac myocytes, MMP-2 impairs β1 integrin-mediated survival signals, such as activation of focal adhesion kinase (FAK), and activates the JNK-dependent mitochondrial death pathway leading to apoptosis.24
Role of Imaging MMP and Apoptosis
Radiolabeled annexin A5 has been successfully employed for the noninvasive detection of macrophage apoptosis in atherosclerotic plaque10,25 and treatment-related changes in apoptosis.3 Similarly, imaging of MMP activity and treatment-related changes in MMP content within the plaque with a radiolabeled MPI has also been successfully demonstrated previously.9 Lipid lowering by dietary modification or HMG-CoA reductase inhibitors, which are associated with an abrogation of apoptosis and a reduction of MMP in plaque are known to stabilize atherosclerotic plaques.
The present study demonstrates the feasibility of molecular imaging of more than a single target by using different tracers. If the candidate targets are interrelated, dual imaging may not offer sufficient incremental value. Nonetheless, molecular imaging helps understand the pathogenesis of a disease better and vice versa; better understanding of a disease process may allow development of improved diagnostic strategies.
The study was supported by National Institutes of Health grant RO1 (HL 078681) provided to Jagat Narula, MD. He received AA5 from PharmaTarget Inc., Maastricht, Netherlands, MPI from Lantheus Imaging Inc., N. Billerica, MA, and fluvastatin from Novartis Pharma and Tanabe Company, Japan.
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