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1.  High resolution tumor targeting in living mice by means of multispectral optoacoustic tomography 
EJNMMI Research  2012;2:14.
Tumor targeting is of high clinical and biological relevance, and major efforts have been made to develop molecular imaging technologies for visualization of the disease markers in tissue. Of particular interest is apoptosis which has a profound role within tumor development and has significant effect on cancer malignancy.
Herein, we report on targeting of phosphatidylserine-exposing cells within live tumor allograft models using a synthetic near infrared zinc(II)-dipicolylamine probe. Visualization of the probe biodistribution is performed with whole body multispectral optoacoustic tomography (MSOT) system and subsequently compared to results attained by planar and tomographic fluorescence imaging systems.
Compared to whole body optical visualization methods, MSOT attains remarkably better imaging capacity by delivering high-resolution scans of both disease morphology and molecular function in real time. Enhanced resolution of MSOT clearly showed that the probe mainly localizes in the vessels surrounding the tumor, suggesting that its tumor selectivity is gained by targeting the phosphatidylserine exposed on the surface of tumor vessels.
The current study demonstrates the high potential of MSOT to broadly impact the fields of tumor diagnostics and preclinical drug development.
PMCID: PMC3337810  PMID: 22464315
Optoacoustic imaging; Tumor targeting; Molecular imaging; Phosphatidylserine targeting
2.  Multispectral optoacoustic tomography of myocardial infarction 
Photoacoustics  2012;1(1):3-8.
► Multispectral optoacoustics enables in vivo preclinical imaging of heart infarct. ► The spatial resolution is superior to previous deep-tissue optical techniques. ► Molecular contrast is provided by an exogenous inflammation-targeted agent. ► Endogenous contrast provides an anatomical reference for the molecular signal.
To investigate the feasibility of a high resolution optical imaging strategy for myocardial infarction.
Near-infrared approaches to imaging cardiovascular disease enable visualization of disease-associated biological processes in vivo. However, even at the scale of small animals, the strong scattering of light prevents high resolution imaging after the first 1–2 mm of tissue, leading to degraded signal localization.
Multispectral optoacoustic tomography (MSOT) was used to non-invasively image myocardial infarction (MI) in a murine model of coronary artery ligation at resolutions not possible with current deep-tissue optical imaging methods. Post-MI imaging was based on resolving the spectral absorption signature of a dendritic polyglycerol sulfate-based (dPGS) near-infrared imaging agent targeted to P- and L-selectin.
In vivo imaging succeeded in detection of the agent in the injured myocardium after intravenous injection. The high anatomic resolution (<200 μm) achieved by the described method allowed signals originating in the infarcted heart to be distinguished from uptake in adjacent regions. Histological analysis found dPGS signal in infarcted areas, originating from leukocytes and endothelial cells.
MSOT imaging of myocardial infarction provides non-invasive visualization of optical contrast with a high spatial resolution that is not degraded by the scattering of light.
PMCID: PMC4182822  PMID: 25327410
Myocardial infarction; Optical imaging; Optoacoustic imaging
3.  Functional optoacoustic human angiography with handheld video rate three dimensional scanner☆ 
Photoacoustics  2013;1(3-4):68-73.
Optoacoustic imaging provides a unique combination of high optical contrast and excellent spatial resolution, making it ideal for simultaneous imaging of tissue anatomy as well as functional and molecular contrast in deep optically opaque tissues. We report on development of a portable clinical system for three-dimensional optoacoustic visualization of deep human tissues at video rate. Studies in human volunteers have demonstrated powerful performance in delivering high resolution volumetric multispectral optoacoustic tomography (vMSOT) images of tissue morphology and function, such as blood oxygenation parameters, in real time. Whilst most imaging modalities currently in clinical use are not able to deliver volumetric data with comparable time resolution, the presented imaging approach holds promise to attain new diagnostic and treatment monitoring value for multiple indications, such as cardiovascular and peripheral vascular disease, disorders related to the lymphatic system, breast lesions, arthritis and inflammation.
PMCID: PMC4134902  PMID: 25302151
Optoacoustic imaging; Cardiovascular diagnostics; Functional and molecular imaging
4.  Semi-quantitative Multispectral Optoacoustic Tomography (MSOT) for volumetric PK imaging of gastric emptying 
Photoacoustics  2014;2(3):103-110.
Graphical abstract
A common side effect of medication is gastrointestinal intolerance. Symptoms can include reduced appetite, diarrhea, constipation, GI inflammation, nausea and vomiting. Such effects often have a dramatic impact on compliance with a treatment regimen. Therefore, characterization of GI tolerance is an important step when establishing a novel therapeutic approach.
In this study, Multispectral Optoacoustic Tomography (MSOT) is used to monitor gastrointestinal motility by in vivo whole body imaging in mice. MSOT combines high spatial and temporal resolution based on ultrasound detection with strong optical contrast in the near infrared. Animals were given Indocyanine Green (ICG) by oral gavage and imaged by MSOT to observe the fate of ICG in the gastrointestinal tract. Exponential decay of ICG signal was observed in the stomach in good correlation with ex vivo validation. We discuss how kinetic imaging in MSOT allows visualization of parameters unavailable to other imaging methods, both in 2D and 3D.
PMCID: PMC4244636  PMID: 25431754
MSOT, Multispectral Optoacoustic Tomography; ICG, Indocyanine Green; Multispectral Optoacoustic Tomography (MSOT); Photoacoustic imaging; Indocyanine Green; In vivo imaging; Pharmacokinetics; Gastric emptying
5.  Fast Multispectral Optoacoustic Tomography (MSOT) for Dynamic Imaging of Pharmacokinetics and Biodistribution in Multiple Organs 
PLoS ONE  2012;7(1):e30491.
The characterization of pharmacokinetic and biodistribution profiles is an essential step in the development process of new candidate drugs or imaging agents. Simultaneously, the assessment of organ function related to the uptake and clearance of drugs is of great importance. To this end, we demonstrate an imaging platform capable of high-rate characterization of the dynamics of fluorescent agents in multiple organs using multispectral optoacoustic tomography (MSOT). A spatial resolution of approximately 150 µm through mouse cross-sections allowed us to image blood vessels, the kidneys, the liver and the gall bladder. In particular, MSOT was employed to characterize the removal of indocyanine green from the systemic circulation and its time-resolved uptake in the liver and gallbladder. Furthermore, it was possible to track the uptake of a carboxylate dye in separate regions of the kidneys. The results demonstrate the acquisition of agent concentration metrics at rates of 10 samples per second at a single wavelength and 17 s per multispectral sample with 10 signal averages at each of 5 wavelengths. Overall, such imaging performance introduces previously undocumented capabilities of fast, high resolution in vivo imaging of the fate of optical agents for drug discovery and basic biological research.
PMCID: PMC3266258  PMID: 22295087
6.  MMP‐1 and MMP‐9 regulate epidermal growth factor‐dependent collagen loss in human carotid plaque smooth muscle cells 
Physiological Reports  2014;2(2):e00224.
Mechanisms underlying the rupture of atherosclerotic plaque, a crucial factor in the development of myocardial infarction and stroke, are not well defined. Here, we examined the role of epidermal growth factor (EGF)‐mediated matrix metalloproteinases (MMP) on the stability of interstitial collagens in vascular smooth muscle cells (VSMCs) isolated from carotid endarterectomy tissues of symptomatic and asymptomatic patients with carotid stenosis. VSMCs isolated from the carotid plaques of both asymptomatic and symptomatic patients were treated with EGF. The MMP‐9 activity was quantified by gelatin zymography and the analysis of mRNA transcripts and protein for MMP‐9, MMP‐1, EGFR and collagen types I, Col I(α1) and collagen type III, Col III(α1) were analyzed by qPCR and immunofluorescence, respectively. The effect of EGF treatment to increase MMP‐9 activity and mRNA transcripts for MMP‐9, MMP‐1, and EGFR and to decrease mRNA transcripts for Col I(α1) and Col III(α1) was threefold to fourfold greater in VSMCs isolated from the carotid plaques of symptomatic than asymptomatic patients. Inhibitors of EGFR (AG1478) and a small molecule inhibitor of MMP‐9 decreased the MMP9 expression and upregulated Col I(α1) and Col III(α1) in EGF‐treated VSMCs of both groups. Additionally, the magnitude in decreased MMP‐9 mRNA and increased Col I(α1) and Col III(α1) due to knockdown of MMP‐9 gene with siRNA in EGF‐treated VSMCs was significantly greater in the symptomatic group than the asymptomatic group. Thus, a selective blockade of both EGFR and MMP‐9 may be a novel strategy and a promising target for stabilizing vulnerable plaques in patients with carotid stenosis.
This report described the underlying mechanisms by which MMP‐1 and MMP‐9 induced by EFGR activation decreases the interstitial collagens and this could result in plaque instability in patients with carotid stenosis. Thus, selective blockade of EGFR and/or MMP‐9 may be a novel strategy and a promising target to stabilize atherosclerotic plaques and thus decreases morbidity and mortality.
PMCID: PMC3966234  PMID: 24744893
Atherosclerosis; carotid plaques; interstitial collagens; matrix metalloproteinase; vascular smooth muscle cells
7.  Pioglitazone Suppresses Inflammation In Vivo In Murine Carotid Atherosclerosis: Novel Detection by Dual-Target Fluorescence Molecular Imaging 
Anti-inflammatory actions of peroxisome proliferator-activated receptor (PPAR)-γ agonists such as pioglitazone (PIO) may underlie their reported but incompletely understood repression of atherosclerosis. This molecular imaging study investigated the effects of pioglitazone on plaque matrix metalloproteinase (MMP) and macrophage responses in vivo.
Methods and Results
In vitro, pioglitazone suppressed MMP-9 mRNA expression in murine peritoneal macrophages (P<0.05). To assess pioglitazone's effects on plaque inflammation, nondiabetic apoE−/− mice on high-cholesterol diet (HCD) received a MMP-activatable fluorescence imaging agent and a spectrally-distinct macrophage-avid fluorescent nanoparticle. After 24 hours, mice underwent survival dual-target intravital fluorescence microscopy (IVFM) of carotid arterial plaques. These mice were then randomized to HCD or HCD+PIO 0.012% for 8 weeks, followed by a second IVFM study of the same carotid plaque. In the HCD group, in vivo MMP and macrophage target-to-background ratios (TBRs) increased similarly (P<0.01 vs. baseline). In contrast, pioglitazone reduced MMP and macrophage TBRs (P<0.01 vs. HCD). Changes in MMP and macrophage signals correlated strongly (r-values≥0.75). Microscopy demonstrated MMP and macrophage reductions in pioglitazone-treated mice, as well as a PIO-modulated increase in plaque collagen.
Serial optical molecular imaging demonstrates that plaque MMP and macrophage activity in vivo intensify with hypercholesterolemia and are reduced by pioglitazone therapy.
PMCID: PMC3030475  PMID: 20689078
atherosclerosis; pioglitazone; inflammation; molecular imaging; fluorescence
8.  Ligation of Macrophage Fcγ Receptors Recapitulates the Gene Expression Pattern of Vulnerable Human Carotid Plaques 
PLoS ONE  2011;6(7):e21803.
Stroke is a leading cause of death in the United States. As ∼60% of strokes result from carotid plaque rupture, elucidating the mechanisms that underlie vulnerability is critical for therapeutic intervention. We tested the hypothesis that stable and vulnerable human plaques differentially express genes associated with matrix degradation. Examination established that femoral, and the distal region of carotid, plaques were histologically stable while the proximal carotid plaque regions were vulnerable. Quantitative RT-PCR was used to compare expression of 22 genes among these tissues. Distal carotid and femoral gene expression was not significantly different, permitting the distal carotid segments to be used as a paired control for their corresponding proximal regions. Analysis of the paired plaques revealed differences in 16 genes that impact plaque stability: matrix metalloproteinases (MMP, higher in vulnerable), MMP modulators (inhibitors: lower, activators: higher in vulnerable), activating Fc receptors (FcγR, higher in vulnerable) and FcγR signaling molecules (higher in vulnerable). Surprisingly, the relative expression of smooth muscle cell and macrophage markers in the three plaque types was not significantly different, suggesting that macrophage distribution and/or activation state correlates with (in)stability. Immunohistochemistry revealed that macrophages and smooth muscle cells localize to distinct and non-overlapping regions in all plaques. MMP protein localized to macrophage-rich regions. In vitro, treatment of macrophages with immune complexes, but not oxidized low density lipoprotein, C-reactive protein, or TNF-α, induced a gene expression profile similar to that of the vulnerable plaques. That ligation of FcγR recapitulates the pattern of gene expression in vulnerable plaques suggests that the FcγR → macrophage activation pathway may play a greater role in human plaque vulnerability than previously appreciated.
PMCID: PMC3140977  PMID: 21814555
9.  Relationship of MMP-14 and TIMP-3 Expression with Macrophage Activation and Human Atherosclerotic Plaque Vulnerability 
Mediators of Inflammation  2014;2014:276457.
Matrix metalloproteinase-14 (MMP-14) promotes vulnerable plaque morphology in mice, whereas tissue inhibitor of metalloproteinases-3 (TIMP-3) overexpression is protective. MMP-14hi  TIMP-3lo rabbit foam cells are more invasive and more prone to apoptosis than MMP-14lo  TIMP-3hi cells. We investigated the implications of these findings for human atherosclerosis. In vitro generated macrophages and foam-cell macrophages, together with atherosclerotic plaques characterised as unstable or stable, were examined for expression of MMP-14, TIMP-3, and inflammatory markers. Proinflammatory stimuli increased MMP-14 and decreased TIMP-3 mRNA and protein expression in human macrophages. However, conversion to foam-cells with oxidized LDL increased MMP-14 and decreased TIMP-3 protein, independently of inflammatory mediators and partly through posttranscriptional mechanisms. Within atherosclerotic plaques, MMP-14 was prominent in foam-cells with either pro- or anti-inflammatory macrophage markers, whereas TIMP-3 was present in less foamy macrophages and colocalised with CD206. MMP-14 positive macrophages were more abundant whereas TIMP-3 positive macrophages were less abundant in plaques histologically designated as rupture prone. We conclude that foam-cells characterised by high MMP-14 and low TIMP-3 expression are prevalent in rupture-prone atherosclerotic plaques, independent of pro- or anti-inflammatory activation. Therefore reducing MMP-14 activity and increasing that of TIMP-3 could be valid therapeutic approaches to reduce plaque rupture and myocardial infarction.
PMCID: PMC4163186  PMID: 25301980
10.  Rational Design of Matrix Metalloproteinase-13 (MMP-13) Activatable Probes for Enhanced Specificity 
ACS chemical biology  2013;9(2):510-516.
Due to the important roles of matrix metalloproteinases (MMPs) play in tumor invasion and metastasis, various activatable optical probes have been developed to visualize MMP activities in vitro and in vivo. Our recently developed MMP-13 activatable probe, L-MMP-P12, has been successfully applied to image the expression and inhibition of MMPs in a xenografted tumor model (Zhu L et al., Theranostics. 2011;1:18–27). In this study, to further optimize the in vivo behavior of the proteinase activatable probe, we tracked and profiled the metabolites by a high resolution LC/MS system. Two major metabolites that contributed to the fluorescence recovery were identified: One was specifically cleaved between Glycine (G4) and Valine (V5) by MMP, while the other one was generated by non-specific cleavage between Glycine (G7) and Lysine (K8). In order to visualize the MMP activity more accurately and specifically, a new probe D-MMP-P12 was designed by replacing the L-lysine with D-lysine in the MMP substrate sequence. The metabolic profile of the new probe, D-MMP-P12, was further characterized by in vitro enzymatic assay and no non-specific metabolite was found by LC/MS. Our in vivo optical imaging also demonstrated that D-MMP-12 had significantly higher tumor-to-background ratio (TBR, 5.55 ± 0.75) compared with L-MMP-P12 (3.73 ± 0.31) at 2 h post-injection. The improved MMP activatable probe may have the potential for drug screening, tumor diagnosis and therapy response monitoring. Moreover, our research strategy can be further extended to study other protease activatable probes.
PMCID: PMC3944097  PMID: 24266806
Liquid chromatography–mass spectrometry (LC-MS); activatable probe; matrix metalloproteinases (MMPs); metabolite; near-infrared fluorescence imaging
11.  Infrared optical imaging of matrix metalloproteinases (MMPs) up regulation following ischemia reperfusion is ameliorated by hypothermia 
BMC Neuroscience  2012;13:76.
We investigated the use of a new MMP activatable probe MMPSense™ 750 FAST (MMPSense750) for in-vivo visualization of early MMP activity in ischemic stroke. Following middle cerebral artery occlusion (MCAO) optical imaging was performed. Near-infrared (NIR) fluorescent images of MMPSense activation were acquired using an Olympus fluorescent microscope, 1.25x objective, a CCD camera and an appropriate filter cube for detecting the activated probe with peak excitation and emission at 749 and 775 nm, respectively. Images were acquired starting at 2 or 24 hours after reperfusion over the ipsilateral and contralateral cortex before and for 3 hours after, MMPSense750 was injected.
Increased intensities ipsilaterally were observed following MMPSense750 injection with ischemic injury but not in sham animals. There were significant ipsilateral and contralateral differences at 15 minutes (P <0.05) in early ischemic reperfusion and at time 0 in 24 hours post ischemia (P <0.05) which persisted at 180 minutes in both these groups (P <0.01), but not following sham surgery. The increase in ipsilateral signal intensity was attenuated by hypothermia. These observations corresponded with a significant increase in the total MMP-9 protein levels, 5 and 24 hours following ischemia reperfusion (P <0.05) and their reduction by hypothermia.
Matrix-metalloproteinase upregulation in ischemia reperfusion can be imaged acutely in-vivo with NIRF using MMPSense750. Hypothermia attenuated both the optical increase in intensity after MMPSense750 and the increase in MMP-9 protein expression supporting the proof of concept that NIRF imaging using MMPSense can be used to assess potential therapeutic strategies for stroke treatment.
PMCID: PMC3441425  PMID: 22742423
12.  mRNA expression of genes involved in lipid efflux and matrix degradation in occlusive and ectatic atherosclerotic disease 
Journal of Clinical Pathology  2005;58(12):1255-1260.
Background: Atherosclerotic plaque behaviour is influenced by intraplaque inflammation, matrix turnover, and the lipid core volume. Peroxisome proliferator activated receptor γ (PPARγ) modulates atherosclerosis by its anti-inflammatory and anti-protease activity. PPARγ promotes lipid efflux through the liver X receptor α (LXRα) and the ATP binding cassette transporter A1 (ABCA1). Matrix metalloproteinase 9 (MMP-9) and cyclooxygenase 2 (COX-2) are implicated in plaque instability.
Aims: To assess the expression of these genes in occlusive and ectatic atherosclerotic disease to determine the relation between genes involved in lipid efflux and matrix degradation.
Methods: Carotid endarterectomy specimens from 16 patients and aneurysm tissue from 16 patients undergoing abdominal aortic aneurysm repair were used. Inferior mesenteric arteries from colectomy specimens from 12 patients served as controls. Total RNA was extracted from pulverised tissue and reverse transcribed into cDNA. Quantitative real time polymerase chain reaction (PCR) was performed using fluorescently labelled probes for ABCA1, LXRα, PPARγ, COX-2, and MMP-9.
Results: PPARγ expression was significantly lower in both occlusive and ecstatic atherosclerotic disease (p<0.001), whereas LXRα and ABCA1 expression was significantly increased (p<0.01). MMP-9 expression was significantly increased in diseased tissues (p<0.0001), and values were highest in occlusive disease (p<0.01). The increases in ABCA1 and MMP-9 mRNA were significantly correlated in diseased tissues (p<0.01, r = 0.71 and r = 0.78). COX-2 expression was increased in ectatic but low in occlusive disease (p<0.01).
Conclusion: This observational study suggests a role for therapeutic upregulation of PPARγ, which could potentially upregulate lipid efflux through ABCA1 and inhibit matrix degradation through inhibition of MMP-9.
PMCID: PMC1770805  PMID: 16311343
Peroxisome proliferator activated receptor γ; ATP binding cassette transporter A1; cyclooxygenase 2; liver X receptor α; matrix metalloproteinase 9; quantitative real time; polymerase chain reaction; plaque; aneurysm
13.  The activation of the cannabinoid receptor type 2 reduces neutrophilic protease-mediated vulnerability in atherosclerotic plaques 
European Heart Journal  2011;33(7):846-856.
The activation of cannabinoid receptor type 2 (CB2)-mediated pathways might represent a promising anti-atherosclerotic treatment. Here, we investigated the expression of the endocannabinoid system in human carotid plaques and the impact of CB2 pharmacological activation on markers of plaque vulnerability in vivo and in vitro.
Methods and results
The study was conducted using all available residual human carotid tissues (upstream and downstream the blood flow) from our cohort of patients symptomatic (n = 13) or asymptomatic (n = 27) for ischaemic stroke. Intraplaque levels of 2-arachidonoylglycerol, anandamide N-arachidonoylethanolamine, N-palmitoylethanolamine, N-oleoylethanolamine, and their degrading enzymes (fatty acid amide hydrolase and monoacylglycerol lipase) were not different in human plaque portions. In the majority of human samples, CB1 (both mRNA and protein levels) was undetectable. In downstream symptomatic plaques, CB2 protein expression was reduced when compared with asymptomatic patients. In these portions, CB2 levels were inversely correlated (r = −0.4008, P = 0.0170) with matrix metalloprotease (MMP)-9 content and positively (r = 0.3997, P = 0.0174) with collagen. In mouse plaques, CB2 co-localized with neutrophils and MMP-9. Treatment with the selective CB2 agonist JWH-133 was associated with the reduction in MMP-9 content in aortic root and carotid plaques. In vitro, pre-incubation with JWH-133 reduced tumour necrosis factor (TNF)-α-mediated release of MMP-9. This effect was associated with the reduction in TNF-α-induced ERK1/2 phosphorylation in human neutrophils.
Cannabinoid receptor type 2 receptor is down-regulated in unstable human carotid plaques. Since CB2 activation prevents neutrophil release of MMP-9 in vivo and in vitro, this treatment strategy might selectively reduce carotid vulnerability in humans.
PMCID: PMC3345556  PMID: 22112961
Carotid arteries; Metalloproteinase-9; Neutrophils
14.  Plaque Rupture is a Determinant of Vascular Events in Carotid Artery Atherosclerotic Disease: Involvement of Matrix Metalloproteinases 2 and 9 
Background and Purpose
Unstable carotid atherosclerotic plaques are characterized by cap rupture, leading to thromboembolism and stroke. Matrix metalloproteinases (MMPs) have been implicated in the progression of atherosclerosis and plaque rupture. The aim of this study was to assess the relationship between the expressions of MMP-2 and MMP-9 and carotid plaque instability.
Eighty atherosclerotic plaques were collected from 74 patients undergoing carotid endarterectomy. Clinical information was obtained from each patient, and plaque morphology was examined at the macroscopic and microscopic levels. The immunohistochemical expressions of MMPs were graded using semiquantitative scales.
Macroscopic ulceration (84.6% versus 63.4%, p=0.042) and microscopic cap rupture (79.5% versus 51.2%, p=0.010) were more common in symptomatic than in asymptomatic patients. Immunoreactivities of MMP-2 and MMP-9 were increased in 40 and 36 atheromatous plaques, respectively. Macroscopic ulceration was strongly correlated with the expressions of MMP-2 (p<0.001) and MMP-9 (p=0.001). There were significant correlations between increased MMP-2 expression and cap rupture (p=0.002), intraplaque hemorrhage (p=0.039), and a thin fibrous cap (p=0.002), and between increased MMP-9 expression and cap rupture (p=0.010) and a large lipid core (p=0.013).
Plaque rupture was significantly associated with the development of vascular events in carotid atherosclerotic disease. MMP-2 and MMP-9 are strongly correlated with plaque instability.
PMCID: PMC3131541  PMID: 21779294
metalloproteinase; carotid plaque; instability
15.  The Relationships Between Regional Arterial Inflammation, Calcification, Risk Factors and Biomarkers – A Prospective FDG PET/CT Imaging Study 
Fluorodeoxyglucose positron emission tomography (FDG PET) imaging of atherosclerosis has been used to quantify plaque inflammation and to measure the effect of plaque stabilizing drugs. Here we explore how atherosclerotic plaque inflammation varies across arterial territories and how it relates to arterial calcification. We also test the hypotheses that the degree of local arterial inflammation measured by PET is correlated with the extent of systemic inflammation and presence of risk factors for vascular disease.
Methods and Results
Forty-one subjects underwent vascular PET/CT imaging with FDG. All had either vascular disease or multiple risk factors for it. Forty subjects underwent carotid imaging, twenty-seven underwent aortic, twenty-four iliac and thirteen femoral imaging. Thirty-three subjects had a panel of biomarkers analyzed.
We found strong associations between FDG uptake in neighboring arteries (left vs. right carotid r=0.91, p<0.001, ascending aorta vs. aortic arch r=0.88, p<0.001). Calcification and inflammation rarely overlapped within arteries – carotid artery FDG uptake vs. calcium score r=−0.42, p=0.03). Carotid artery FDG uptake was greater in those with a history of coronary artery disease (target to background ratio (TBR) 1.83 vs. 1.61, p<0.01), and in males vs. females (TBR 1.83 vs. 1.63, p<0.05). Similar findings were also noted in the aorta and iliac arteries. Subjects with the highest levels of FDG uptake also had the greatest concentrations of inflammatory biomarkers: descending aorta TBR vs. matrix metalloproteinase 3 (MMP 3): r=0.53, p=0.01 and carotid TBR vs. MMP 9: r=0.50, p=0.01. Non-significant positive trends were seen between FDG uptake and levels of interleukin 18, fibrinogen and C-reactive protein. Finally, we found that the atheroprotective biomarker adiponectin was negatively correlated with the degree of arterial inflammation in the descending aorta: r=−0.49, p=0.03).
This study shows that FDG PET imaging can increase our knowledge of how atherosclerotic plaque inflammation relates to calcification, serum biomarkers and vascular risk factors. Plaque inflammation and calcification rarely overlap, supporting the theory that calcification represents a late, burnt-out stage of atherosclerosis. Inflammation in one arterial territory is associated with inflammation elsewhere, and the degree of local arterial inflammation is reflected in the blood levels of several circulating biomarkers. We suggest that FDG PET imaging could be used as a surrogate marker of both atherosclerotic disease activity and drug effectiveness. Prospective, event driven studies are now underway to determine the role of this technique in clinical risk prediction.
PMCID: PMC3190196  PMID: 19808576
Atherosclerosis; Imaging; Inflammation; Positron Emission Tomography; Fluorodeoxyglucose; Calcification
16.  Assessment of MMP-9, TIMP-1, and COX-2 in normal tissue and in advanced symptomatic and asymptomatic carotid plaques 
Thrombosis Journal  2011;9:6.
Mature carotid plaques are complex structures, and their histological classification is challenging. The carotid plaques of asymptomatic and symptomatic patients could exhibit identical histological components.
To investigate whether matrix metalloproteinase 9 (MMP-9), tissue inhibitor of MMP (TIMP), and cyclooxygenase-2 (COX-2) have different expression levels in advanced symptomatic carotid plaques, asymptomatic carotid plaques, and normal tissue.
Thirty patients admitted for carotid endarterectomy were selected. Each patient was assigned preoperatively to one of two groups: group I consisted of symptomatic patients (n = 16, 12 males, mean age 66.7 ± 6.8 years), and group II consisted of asymptomatic patients (n = 14, 8 males, mean age 67.6 ± 6.81 years). Nine normal carotid arteries were used as control. Tissue specimens were analyzed for fibromuscular, lipid and calcium contents. The expressions of MMP-9, TIMP-1 and COX-2 in each plaque were quantified.
Fifty-eight percent of all carotid plaques were classified as Type VI according to the American Heart Association Committee on Vascular Lesions. The control carotid arteries all were classified as Type III. The median percentage of fibromuscular tissue was significantly greater in group II compared to group I (p < 0.05). The median percentage of lipid tissue had a tendency to be greater in group I than in group II (p = 0.057). The percentages of calcification were similar among the two groups. MMP-9 protein expression levels were significantly higher in group II and in the control group when compared with group I (p < 0.001). TIMP-1 expression levels were significantly higher in the control group and in group II when compared to group I, with statistical difference between control group and group I (p = 0.010). COX-2 expression levels did not differ among groups. There was no statistical correlation between MMP-9, COX-2, and TIMP-1 levels and fibrous tissue.
MMP-9 and TIMP-1 are present in all stages of atherosclerotic plaque progression, from normal tissue to advanced lesions. When sections of a plaque are analyzed without preselection, MMP-9 concentration is higher in normal tissues and asymptomatic surgical specimens than in symptomatic specimens, and TIMP-1 concentration is higher in normal tissue than in symptomatic specimens.
PMCID: PMC3076223  PMID: 21457581
17.  Matrix Metalloproteinase 7 Is Associated with Symptomatic Lesions and Adverse Events in Patients with Carotid Atherosclerosis 
PLoS ONE  2014;9(1):e84935.
Atherosclerosis is a major cause of cerebrovascular disease. Matrix metalloproteinases (MMPs) play an important role in matrix degradation within the atherosclerotic lesion leading to plaque destabilization and ischemic stroke. We hypothesized that MMP-7 could be involved in this process.
Plasma levels of MMP-7 were measured in 182 consecutive patients with moderate (50–69%) or severe (≥70%) internal carotid artery stenosis, and in 23 healthy controls. The mRNA levels of MMP-7 were measured in atherosclerotic carotid plaques with different symptomatology, and based on its localization to macrophages, the in vitro regulation of MMP-7 in primary monocytes was examined.
Our major findings were (i) Patients with carotid atherosclerosis had markedly increased plasma levels of MMP-7 compared to healthy controls, with particularly high levels in patients with recent symptoms (i.e., within the last 2 months). (ii) A similar pattern was found within carotid plaques with markedly higher mRNA levels of MMP-7 than in non-atherosclerotic vessels. Particularly high protein levels of MMP-7 levels were found in those with the most recent symptoms. (iii) Immunhistochemistry showed that MMP-7 was localized to macrophages, and in vitro studies in primary monocytes showed that the inflammatory cytokine tumor necrosis factor-α in combination with hypoxia and oxidized LDL markedly increased MMP-7 expression. (iv) During the follow-up of patients with carotid atherosclerosis, high plasma levels of MMP-7 were independently associated with total mortality.
Our findings suggest that MMP-7 could contribute to plaque instability in carotid atherosclerosis, potentially involving macrophage-related mechanisms.
PMCID: PMC3882279  PMID: 24400123
18.  Caveolin-1 Influences Vascular Protease Activity and Is a Potential Stabilizing Factor in Human Atherosclerotic Disease 
PLoS ONE  2008;3(7):e2612.
Caveolin-1 (Cav-1) is a regulatory protein of the arterial wall, but its role in human atherosclerosis remains unknown. We have studied the relationships between Cav-1 abundance, atherosclerotic plaque characteristics and clinical manisfestations of atherosclerotic disease.We determined Cav-1 expression by western blotting in atherosclerotic plaques harvested from 378 subjects that underwent carotid endarterectomy. Cav-1 levels were significantly lower in carotid plaques than non-atherosclerotic vascular specimens. Low Cav-1 expression was associated with features of plaque instability such as large lipid core, thrombus formation, macrophage infiltration, high IL-6, IL-8 levels and elevated MMP-9 activity. Clinically, a down-regulation of Cav-1 was observed in plaques obtained from men, patients with a history of myocardial infarction and restenotic lesions. Cav-1 levels above the median were associated with absence of new vascular events within 30 days after surgery [0% vs. 4%] and a trend towards lower incidence of new cardiovascular events during longer follow-up. Consistent with these clinical data, Cav-1 null mice revealed elevated intimal hyperplasia response following arterial injury that was significantly attenuated after MMP inhibition. Recombinant peptides mimicking Cav-1 scaffolding domain (Cavtratin) reduced gelatinase activity in cultured porcine arteries and impaired MMP-9 activity and COX-2 in LPS-challenged macrophages. Administration of Cavtratin strongly impaired flow-induced expansive remodeling in mice.This is the first study that identifies Cav-1 as a novel potential stabilizing factor in human atherosclerosis. Our findings support the hypothesis that local down-regulation of Cav-1 in atherosclerotic lesions contributes to plaque formation and/or instability accelerating the occurrence of adverse clinical outcomes. Therefore, given the large number of patients studied, we believe that Cav-1 may be considered as a novel target in the prevention of human atherosclerotic disease and the loss of Cav-1 may be a novel biomarker of vulnerable plaque with prognostic value.
PMCID: PMC2432041  PMID: 18596970
19.  Active synovial matrix metalloproteinase-2 is associated with radiographic erosions in patients with early synovitis 
Arthritis Research  2000;2(2):145-153.
Serum and synovial tissue expression of the matrix metalloproteinase (MMP)-2 and -9 and their molecular regulators, MMP-14 and TIMP-2 was examined in 28 patients with inflammatory early synovitis and 4 healthy volunteers and correlated with the presence of erosions in the patients. Immunohistological staining of MMP-2, MMP-14 and TIMP-2 localized to corresponding areas in the synovial lining layer and was almost absent in normal synovium. Patients with radiographic erosions had significantly higher levels of active MMP-2 than patients with no erosions, suggesting that activated MMP-2 levels in synovial tissue may be a marker for a more aggressive synovial lesion.
In cancer the gelatinases [matrix metalloproteinase (MMP)-2 and MMP-9] have been shown to be associated with tissue invasion and metastatic disease. In patients with inflammatory arthritis the gelatinases are expressed in the synovial membrane, and have been implicated in synovial tissue invasion into adjacent cartilage and bone. It is hypothesized that an imbalance between the activators and inhibitors of the gelatinases results in higher levels of activity, enhanced local proteolysis, and bone erosion.
To determine whether the expression and activity levels of MMP-2 and MMP-9, and their regulators MMP-14 and tissue inhibitor of metalloproteinase (TIMP), are associated with early erosion formation in patients with synovitis of recent onset.
Patients and method:
A subset of 66 patients was selected from a larger early synovitis cohort on the basis of tissue availability for the study of synovial tissue and serum gelatinase expression. Patients with peripheral joint synovitis of less than 1 years' duration were evaluated clinically and serologically on four visits over a period of 12 months. At the initial visit, patients underwent a synovial tissue biopsy of one swollen joint, and patients had radiographic evaluation of hands and feet initially and at 1year. Serum MMP-1, MMP-2, MMP-9, MMP-14, and TIMP-1 and TIMP-2 levels were determined, and synovial tissue was examined by immunohistology for the expression of MMP-2 and MMP-9, and their molecular regulators. Gelatinolytic activity for MMP-2 and MMP-9 was quantified using a sensitive, tissue-based gel zymography technique. Four healthy individuals underwent closed synovial biopsy and their synovial tissues were similarly analyzed.
Of the 66 patients studied, 45 fulfilled American College of Rheumatology criteria for rheumatoid arthritis (RA), with 32 (71%) being rheumatoid factor positive. Of the 21 non-RA patients, seven had a spondylarthropathy and 14 had undifferentiated arthritis. Radiographically, 12 of the RA patients had erosions at multiple sites by 1 year, whereas none of the non-RA patients had developed erosive disease of this extent. In the tissue, latent MMP-2 was widely expressed in the synovial lining layer and in areas of stromal proliferation in the sublining layer and stroma, whereas MMP-9 was expressed more sparsely and focally. MMP-14, TIMP-2, and MMP-2 were all detected in similar areas of the lining layer on consecutive histologic sections. Tissue expression of MMP-14, the activator for pro-MMP-2, was significantly higher in RA than in non-RA patients (8.4 ± 5 versus 3.7 ± 4 cells/high-power field; P = 0.009). In contrast, the expression of TIMP-2, an inhibitor of MMP-2, was lower in the RA than in the non-RA samples (25 ± 12 versus 39 ± 9 cells/high-power field; P = 0.01). Synovial tissue expressions of MMP-2, MMP-14, and TIMP-2 were virtually undetectable in normal synovial tissue samples. The synovial tissue samples of patients with erosive disease had significantly higher levels of active MMP-2 than did those of patients without erosions (Fig. 1). Tissue expression of MMP-2 and MMP-9, however, did not correlate with the serum levels of these enzymes.
With the exception of serum MMP-2, which was not elevated over normal, serum levels of all of the other MMPs and TIMPs were elevated to varying degrees, and were not predictive of erosive disease. Interestingly, MMP-1 and C-reactive protein, both of which were associated with the presence of erosions, were positively correlated with each other (r = 0.42; P < 0.001).
MMP-2 and MMP-9 are thought to play an important role in the evolution of joint erosions in patients with an inflammatory arthritis. Most studies have concentrated on the contribution of MMP-9 to the synovitis, because synovial fluid and serum MMP-9 levels are markedly increased in inflammatory arthropathies. Previously reported serum levels of MMP-9 have varied widely. In the present sample of patients with synovitis of recent onset, serum MMP-9 levels were elevated in only 21%. Moreover, these elevations were not specific for RA, the tissue expression of MMP-9 was focal, and the levels of MMP-9 activity were not well correlated with early erosions. Although serum MMP-2 levels were not of prognostic value, high synovial tissue levels of MMP-2 activity were significantly correlated with the presence of early erosions. This may reflect augmented activation of MMP-2 by the relatively high levels of MMP-14 and low levels of TIMP-2 seen in these tissues. We were able to localize the components of this trimolecular complex to the synovial lining layer in consecutive tissue sections, a finding that is consistent with their colocalization.
In conclusion, we have provided evidence that active MMP-2 complexes are detectable in the inflamed RA synovium and may be involved in the development of early bony erosions. These results suggest that strategies to inhibit the activation of MMP-2 may have the potential for retarding or preventing early erosions in patients with inflammatory arthritis.
PMCID: PMC17808  PMID: 11062605
early synovitis; erosion; metalloproteinase; matrix metalloproteinase-2; rheumatoid arthritis
20.  Association of Circulating Matrix Metalloproteinases with Carotid Artery Characteristics: The ARIC Carotid MRI Study 
To examine the relationship of plasma levels of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinase-1 (TIMP-1) with carotid artery characteristics measured by magnetic resonance imaging (MRI) in a cross-sectional investigation among Atherosclerosis Risk in Communities (ARIC) Carotid MRI Study participants.
Methods and Results
A stratified random sample was recruited based on intima-media thickness (IMT) from a previous ultrasound examination. A high-resolution gadolinium-enhanced MRI exam of the carotid artery was performed in 2004–2005 on 1,901 ARIC cohort participants. Multiple carotid wall characteristics including wall thickness, lumen area, calcium area, lipid core and fibrous cap measures were evaluated for associations with plasma MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9 and TIMP-1.
Plasma MMP-1, MMP-3, and MMP-7 were significantly higher among participants in the high IMT group compared to those in the low IMT group. Normalized wall index was independently associated with MMP-3, MMP-7, and TIMP-1. MMP-7 was positively associated with carotid calcification. Mean fibrous cap thickness was significantly higher in individuals with elevated TIMP-1 levels. In addition, TIMP-1 was positively associated with measures of lipid core.
Circulating levels of specific MMPs and TIMP-1 were associated with carotid wall remodeling and structural changes related to plaque burden in the elderly.
PMCID: PMC2860383  PMID: 20167662
Atherosclerosis; Carotid; MRI; MMP; TIMP-1; IMT; Luminex; Multianalyte Profiling
21.  Plaque Rupture Complications in Murine Atherosclerotic Vein Grafts Can Be Prevented by TIMP-1 Overexpression 
PLoS ONE  2012;7(10):e47134.
The current study describes the incidence and phenotype of plaque rupture complications in murine vein grafts. Since matrix metalloproteinases (MMPs) are highly involved in atherosclerotic plaque vulnerability and plaque rupture, we hypothesized that this model can be validated by overexpression of the MMP inhibitor TIMP-1. First we studied 47 vein grafts in hypercholesterolemic ApoE3*Leiden mice for the incidence of plaque complications. In 79% of these grafts, extensive lesions with plaque rupture complications like dissections, intraplaque hemorrhages or erosions with intramural thrombi were found. Next, in vivo Near-InfraRed-Fluorescence imaging demonstrated that electroporation mediated TIMP-1-overexpression reduced local MMP activity in vein grafts by 73% (p<0.01). This led to a 40% reduction in lesion-size after 28d (p = 0.01) and a more stable lesion phenotype with significant more smooth muscle cells (135%), collagen (47%) and significant less macrophages (44%) and fibrin (55%) than controls. More importantly, lesions in the TIMP-1 group showed a 90% reduction of plaque complications (10/18 of control mice showed plaque complications versus 1/18 in TIMP-1 treated mice). Murine vein grafts are a relevant spontaneous model to study plaque stability and subsequent hemorrhagic complications, resulting in plaque instability. Moreover, inhibition of MMPs by TIMP-1-overexpression resulted in decreased plaque progression, increased stabilization and decreased plaque rupture complications in murine vein grafts.
PMCID: PMC3469549  PMID: 23071737
22.  Optical Tomography of MMP Activity Allows a Sensitive Noninvasive Characterization of the Invasiveness and Angiogenesis of SCC Xenografts12 
Neoplasia (New York, N.Y.)  2014;16(3):235-246.e1.
For improved tumor staging and therapy control, imaging biomarkers are of great interest allowing a noninvasive characterization of invasiveness. In squamous epithelial skin and cervix lesions, transition to invasive stages is associated with enhanced matrix metalloproteinase (MMP) activity, increased angiogenesis, and worsened prognosis. Thus, we investigated MMP activity as imaging biomarker of invasiveness and the potential of optical tomography in characterizing the angiogenic and invasive behavior of skin squamous cell carcinoma (SCC) xenografts. MMP activity was measured in vivo in HaCaT-ras A-5RT3 tumors at different angiogenic and invasive stages (onset of angiogenesis, intermediate and highly angiogenic, invasive stage) and after 1 week of sunitinib treatment by fluorescence molecular tomography–microcomputed tomography imaging using an activatable probe. Treatment response was additionally assessed morphologically by optical coherence tomography (OCT). In vivo MMP activity significantly differed between the groups, revealing highest levels in the highly angiogenic, invasive tumors that were confirmed by immunohistochemistry. At the onset of angiogenesis with lowest MMP activity, fibroblasts were detected in the MMP-positive areas, whereas macrophages were absent. Accumulation of both cell types occurred in both invasive groups, again to a significantly higher degree at the most invasive and angiogenic stage. Sunitinib treatment significantly reduced the MMP activity and accumulation of fibroblasts and macrophages and blocked tumor invasion that was additionally visualized by OCT. Human cervical SCCs also showed high MMP activity and a similar stromal composition as the HaCaT xenografts, whereas normal tissue was negative. This study strongly suggests MMP activity as imaging biomarker and demonstrates the high sensitivity of optical tomography in determining tumor invasiveness that can morphologically be supported by OCT.
PMCID: PMC4094793  PMID: 24784000
SCC, squamous cell carcinoma; MMP, matrix metalloproteinase; ECM, extracellular matrix; FMT, fluorescence molecular tomography; μCT, micro-computed tomography; OCT, optical coherence tomography; i.d., intradermal; i.p., intraperitoneal; i.v., intravenous; s.c., subcutaneous; SMA, smooth muscle actin; VEGF, vascular endothelial growth factor; VEGFR2, vascular endothelial growth factor receptor number 2; PK, pan-keratin
23.  Molecular Photoacoustic Imaging of Follicular Thyroid Carcinoma 
To evaluate the potential of targeted photoacoustic imaging as a non-invasive method for detection of follicular thyroid carcinoma.
Experimental Design
We determined the presence and activity of two members of matrix metalloproteinase family (MMP), MMP-2 and MMP-9, suggested as biomarkers for malignant thyroid lesions, in FTC133 thyroid tumors subcutaneously implanted in nude mice. The imaging agent used to visualize tumors was MMP activatable photoacoustic probe, Alexa750-CXeeeeXPLGLAGrrrrrXK-BHQ3. Cleavage of the MMP activatable agent was imaged after intratumoral and intravenous injections in living mice optically, observing the increase in Alexa750 fluorescence, and photoacoustically, using a dual wavelength imaging method.
Active forms of both MMP2 and MMP-9 enzymes were found in FTC133 tumor homogenates, with MMP-9 detected in greater amounts. The molecular imaging agent was determined to be activated by both enzymes in vitro, with MMP-9 being more efficient in this regard. Both optical and photoacoustic imaging showed significantly higher signal in tumors of mice injected with the active agent than in tumors injected with the control, non-activatable, agent.
With the combination of high spatial resolution and signal specificity, targeted photoacoustic imaging holds great promise as a noninvasive method for early diagnosis of follicular thyroid carcinomas.
PMCID: PMC3602312  PMID: 23349314
Photoacoustic Molecular Imaging; Thyroid follicular cancer; Activatable Photoacoustic Probe; Matrix Metalloproteinase; Optical Imaging
24.  Doxycycline Stabilizes Vulnerable Plaque via Inhibiting Matrix Metalloproteinases and Attenuating Inflammation in Rabbits 
PLoS ONE  2012;7(6):e39695.
Enhanced matrix metalloproteinases (MMPs) activity is implicated in the process of atherosclerotic plaque instability. We hypothesized that doxycycline, a broad MMPs inhibitor, was as effective as simvastatin in reducing the incidence of plaque disruption. Thirty rabbits underwent aortic balloon injury and were fed a high-fat diet for 20 weeks. At the end of week 8, the rabbits were divided into three groups for 12-week treatment: a doxycycline-treated group that received oral doxycycline at a dose of 10 mg/kg/d, a simvastatin-treated group that received oral simvastatin at a dose of 5 mg/kg/d, and a control group that received no treatment. At the end of week 20, pharmacological triggering was performed to induce plaque rupture. Biochemical, ultrasonographic, pathologic, immunohistochemical and mRNA expression studies were performed. The results showed that oral administration of doxycycline resulted in a significant increase in the thickness of the fibrous cap of the aortic plaque whereas there was a substantial reduction of MMPs expression, local and systemic inflammation, and aortic plaque vulnerability. The incidence of plaque rupture with either treatment (0% for both) was significantly lower than that for controls (56.0%, P<0.05). There was no significant difference between doxycycline-treated group and simvastatin-treated group in any serological, ultrasonographic, pathologic, immunohistochemical and mRNA expression measurement except for the serum lipid levels that were higher with doxycycline than with simvastatin treatment. In conclusion, doxycycline at a common antimicrobial dose stabilizes atherosclerotic lesions via inhibiting matrix metalloproteinases and attenuating inflammation in a rabbit model of vulnerable plaque. These effects were similar to a large dose of simvastatin and independent of serum lipid levels.
PMCID: PMC3380900  PMID: 22737253
25.  Following the Trajectory of Osteoarthritis Development Through Serial Near Infrared Fluorescence Imaging of MMP Activities 
A major hurdle in osteoarthritis (OA) research is the lack of sensitive detection and monitoring methods. It is hypothesized that proteases, such as matrix metalloproteinases (MMPs), are upregulated at early stages of OA development. The aim of this study was to investigate if a near infrared fluorescence (NIRF) probe activated by MMPs could visualize in vivo OA progression starting from its early stages.
Using an MMP activatable NIRF probe (MMPSense680), we assessed the upregulation of MMP activity in vitro by incubating human chondrocytes with the pro-inflammatory cytokine IL-1β. MMP activity was then evaluated in vivo serially in a chronic, injury-induced OA mouse model. For tracking MMP activity over time, mice were imaged 1 – 8 weeks post OA inducing surgery. Imaging results were correlated with histology.
In vitro studies confirmed that NIRF imaging could identify enhanced MMP activity in IL-1β-treated human chondrocytes. In vivo imaging showed significantly higher fluorescent intensity in OA knees compared to sham knees (control) of the same mice. Additionally, the total emitted fluorescence intensity steadily increased over the entire course of OA progression that was examined. NIRF imaging results correlated with histological analysis, which showed an increase in articular cartilage structural damage over time.
Imaging of MMP activity in an OA mouse model provided sensitive and consistent visualization of OA progression, beginning from the early stages of OA. In addition to facilitating the preclinical study of OA modulators, this approach has the potential for future human translation.
PMCID: PMC4312249  PMID: 25385707

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