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.
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.
Optoacoustic imaging; Tumor targeting; Molecular imaging; Phosphatidylserine targeting
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.
atherosclerosis; pioglitazone; inflammation; molecular imaging; fluorescence
The progression of atherosclerosis involves complex changes in the structure, composition and biology of the artery wall. Currently, only anatomical plaque burden is routinely characterized in living patients, whereas compositional and biological changes are mostly inaccessible. However, anatomical imaging alone has proven to be insufficient for accurate diagnostics of the disease. Multispectral optoacoustic tomography offers complementary data to anatomical methods and is capable of imaging both tissue composition and, via the use of molecular markers, the biological activity therein. In this paper we review recent progress in multispectral optoacoustic tomography imaging of atherosclerosis with specific emphasis on intravascular applications. The potential capabilities of multispectral optoacoustic tomography are compared with those of established intravascular imaging techniques and current challenges on the road towards a clinically viable imaging modality are discussed.
atherosclerosis; intravascular imaging; molecular imaging; multispectral imaging; optical imaging; optoacoustic imaging; photoacoustic imaging; ultrasound
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.
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.
A significant proportion of colorectal adenomas, in particular those that lack an elevated growth component, continue to escape detection during endoscopic surveillance. Elevation of the activity of matrix metalloproteinases (MMPs), a large family of zinc endopeptidases, in adenomas serves as a biomarker of early tumorigenesis. The goal of this study was to assess the feasibility of using a newly developed near-infrared bioactivatable probe (MMPSense 680) that reports the activity of a broad array of MMP isoforms to detect early colorectal adenomas. Adenomatous polyposis coli (Apc)+/Min-FCCC mice that spontaneously develop multiple colorectal adenomas were injected with MMPSense 680, and the colons were imaged in an IVIS Spectrum system ex vivo. Image analyses were correlated with histopathologic findings for all regions of interest (ROIs). The biochemical basis of fluorescent signal was investigated by immunohistochemical staining of MMP-7 and -9. A strong correlation (Kendall = 0.80) was observed between a positive signal and the presence of pathologically confirmed colonic adenomas; 92.9% of the 350 ROIs evaluated were classified correctly. The correlation between two independent observers was 0.87. MMP-7 expression was localized to epithelial cells of adenomas and microadenomas, whereas staining of MMP-9 was found in infiltrating polymorphonuclear leukocytes within the adenomas. MMPSense 680 identifies colorectal adenomas, both polypoid and nonpolypoid, in Apc+/Min-FCCC mice with high specificity. Use of this fluorescent probe in combination with colonoscopy could aid in preventing colorectal neoplasias by providing new opportunities for early detection and therapeutic intervention.
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.
Matrix metalloproteinase (MMP) -2 and -9 play important roles in the progression of atherosclerosis. This study aims to determine whether MMP-2 and -9 content in the fibrotic caps of atherosclerotic plaque is correlated with plaque autofluorescence. A time-resolved laser-induced fluorescence spectroscopy (TR-LIFS) system was used to measure the autofluorescence and assess the biochemical composition of human plaques obtained from carotid endarterectomy. Results presented here demonstrate for the first time the ability to characterize the biochemical composition as it relates to MMP-2 and -9 content in the atherosclerotic plaque cap using a label-free imaging technique implemented with a fiberoptic TR-LIFS system.
Matrix metalloproteinases; time-resolved fluorescence spectroscopy; atherosclerosis; carotid plaque
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.
metalloproteinase; carotid plaque; instability
Plaque rupture is the main cause of acute myocardial infarction and stroke. Atherosclerotic plaques have been described to be vulnerable and more prone to rupture when they are characterized by thin, highly inflamed, and collagen-poor fibrous caps and contain elevated levels of proteases, including metalloproteinases (MMPs). Initiation of collagen breakdown in plaques requires interstitial collagenases, a MMP subfamily consisting of MMP-1, MMP-8, and MMP-13. Previous reports demonstrated that MMP-1 and MMP-13 might be overexpressed in both human and experimental atherosclerosis. Since neutrophils have been only recently reported in atherosclerotic plaques, the role of MMP-8 (formerly known as “neutrophil collagenase”) was only marginally evaluated. In this paper, we will update and comment on evidence of the most relevant regulatory pathways and activities mediated by MMP-8 in atherogenesis.
Detection of atherosclerotic plaque vulnerability has critical clinical implications for avoiding sudden death in patients with high risk of plaque rupture. We report on multimodality imaging of ex-vivo human carotid plaque samples using a system that integrates fluorescence lifetime imaging (FLIM), ultrasonic backscatter microscopy (UBM), and photoacoustic imaging (PAI). Biochemical composition is differentiated with a high temporal resolution and sensitivity at the surface of the plaque by the FLIM subsystem. 3D microanatomy of the whole plaque is reconstructed by the UBM. Functional imaging associated with optical absorption contrast is evaluated from the PAI component. Simultaneous recordings of the optical, ultrasonic, and photoacoustic data present a wealth of complementary information concerning the plaque composition, structure, and function that are related to plaque vulnerability. This approach is expected to improve our ability to study atherosclerotic plaques. The multimodal system presented here can be translated into a catheter based intraluminal system for future clinical studies.
(170.6510) Spectroscopy, tissue diagnostics; (300.6500) Spectroscopy, time-resolved; (110.7170) Ultrasound; (170.5120) Photoacoustic imaging; (170.6935) Tissue characterization
Osteoarthritis (OA) is a degenerative disease starting with key molecular events that ultimately lead to the breakdown of the cartilage. The purpose of this study is to use two imaging methods that are sensitive to molecular and macromolecular changes in OA to better characterize the disease process in human osteoarthritic cartilage.
Human femoral condyles were collected from patients diagnosed with severe OA during total knee replacement surgeries. T1ρ and T2 magnetic resonance measurements were obtained using a 3-Tesla whole body scanner to assess macromolecular changes in the damaged cartilage matrix. Optical imaging was performed on specimens treated with MMPSense 680 to assess the matrix metalloproteinase (MMP) activity. A linear regression model was used to assess the correlation of MMP optical data with T1ρ magnetic resonance (MR) measurements. Slices from a representative specimen were removed from regions with high and low optical signals for subsequent histological analysis.
All specimens exhibit high T1ρ and T2 measurements in the range of 48–75 ms and 36–69 ms, respectively. They also show intense photon signals (0.376 to 7.89 × 10−4 cm2) from the activated MMPSense 680 probe, indicative of high MMP activity. The analysis of variance test of the regression model indicates a positive correlation between the MMP optical signal and T1ρ measurements (R2 = 0.8936, P = 0.0044). Histological data also confirmed that regions with high MMP optical signal and intense T1ρ relaxation exhibit severe clefting, abnormal tidemarks, and irregular cellularity.
The high T1ρ and T2 measurements suggest that there is a severe loss of proteoglycans with high water mobility in the damaged cartilage. The intense optical signals found in these specimens indicate the presence of active MMPs, and the positive correlation with T1ρ measurements implicates MMP’s involvement in OA progression, characterized by a severe loss of proteoglycans in the cartilage matrix. The bimodal approach using optical and MR imaging may provide key molecular and macromolecular information of the disease pathway, offering insights toward the development of new tools for the early detection, treatment, and/or prevention of OA.
Osteoarthritis; Optical imaging; MR imaging; Proteoglycans; Matrix metalloproteinase
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.
Radionuclides; SPECT; vulnerable atherosclerotic plaque
Despite primary and secondary prevention, serious cardiovascular events like unstable angina or myocardial infarction still account for one third of all deaths worldwide. Therefore, identifying individual patients with vulnerable plaques at high risk for plaque rupture is a central challenge in cardiovascular medicine. Several non-invasive techniques, such as MRI, multislice computed tomography and electron beam tomography are currently being tested for their ability to identify such patients by morphological criteria. In contrast, molecular imaging techniques use radiolabeled molecules to detect functional aspects in atherosclerotic plaques by visualizing its biological activity. Based upon the knowledge about the pathophysiology of atherosclerosis, various studies in vitro, in vivo and the first clinical trials have used different tracers for plaque imaging studies, including radioactive labelled lipoproteins, components of the coagulation system, cytokines, mediators of the metalloproteinase system, cell adhesion receptors and even whole cells. This review gives an update on the relevant non-invasive plaque imaging approaches using nuclear imaging techniques to detect atherosclerotic vascular lesions.
Plaque imaging; atherosclerosis; radionuclide imaging; vulnerable plaque; thrombogenicity
Matrix metalloproteinases (MMPs) are a family of proteolytic enzymes that are regulated by inflammatory signals to mediate changes in extracellular matrix. Members of the MMP family share sequence homology, act on interstitial protein substrates, acutely participate in inflammatory processes and chronically mediate tissue remodelling. MMPs are important in vascular remodelling, not only in the overall vasculature architecture but also, more importantly, in the advancing atherosclerotic plaque. MMP activation modifies the architecture of the plaque and may directly participate in the process of plaque rupture. MMPs also participate in cardiac remodelling following myocardial infarction and development of dilated cardiomyopathy. Soluble MMPs are now potential biomarkers in delineating cardiovascular risk for plaque rupture and coronary risk. They also constitute innovative direct or indirect targets to modify cardiovascular tissue remodelling in atherosclerosis and heart failure.
Atherosclerosis; Cardiovascular; Heart failure; Matrix metalloproteinases
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.
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
Vulnerable areas of atherosclerotic plaques often contain lipid-laden macrophages and display matrix metalloproteinase activity. We hypothesized that reactive oxygen species released by macrophage-derived foam cells could trigger activation of latent proforms of metalloproteinases in the vascular interstitium. We showed that in vivo generated macrophage foam cells produce superoxide, nitric oxide, and hydrogen peroxide after isolation from hypercholesterolemic rabbits. Effects of these reactive oxygens and that of peroxynitrite, likely to result from simultaneous production of nitric oxide and superoxide, were tested in vitro using metalloproteinases secreted by cultured human vascular smooth muscle cells. Enzymes in culture media or affinity-purified (pro-MMP-2 and MMP-9) were examined by SDS-PAGE zymography, Western blotting, and enzymatic assays. Under the conditions used, incubation with xanthine/xanthine oxidase increased the amount of active gelatinases, while nitric oxide donors had no noticeable effect. Incubation with peroxynitrite resulted in nitration of MMP-2 and endowed it with collagenolytic activity. Hydrogen peroxide treatment showed a catalase-reversible biphasic effect (gelatinase activation at concentrations of 4 microM, inhibition at > or = 10-50 microM). Thus, reactive oxygen species can modulate matrix degradation in areas of high oxidant stress and could therefore contribute to instability of atherosclerotic plaques.
Carotid artery stenting (CAS) is currently a standard procedure to treat severe carotid artery stenosis. This procedure causes mechanical plaque rupture, potentially releasing soluble factors into the circulating blood. The purpose of this study is to clarify whether inflammation factors are released from an atherosclerotic plaque after CAS and whether local release of inflammation factors is associated with periprocedural new ischemic lesions. The study consisted of 35 patients with 40 severely stenotic carotid arteries who underwent CAS. Blood samples were obtained from the aorta before the procedure and from the carotid plaque site just after the procedure. Blood levels of interleukin-6 (IL-6), interleukin-18, matrix metalloproteinase (MMP)-2, and tissue inhibitor of MMP-1 were determined. Diffusion-weighted magnetic resonance imaging was performed before and after the procedure. Among inflammatory markers, IL-6 levels markedly increased at the plaque site in comparison to those at the aorta (P<0.001). The IL-6 levels in the local samples were significantly higher in symptomatic lesions than those in asymptomatic lesions. More importantly, higher local IL-6 levels were associated with the appearance of new ischemic lesions (P=0.003). The association remained significant (P=0.030) after controlling for potential risk factors for CAS. Association of local IL-6 levels and periprocedural new ischemic lesions suggests that massive release from the plaque and entry into the cerebral circulation of IL-6 might be one of important factors on periprocedural complications related to CAS.
carotid artery stenting; complications; embolism; inflammation; interleukins
Genetic variation in matrix metalloproteinase (MMP) promoter regions alters the transcriptional activity of MMPs and has been consistently associated with CHD, presumably through plaque degradation and remodeling. We examined the association of MMP promoter variation with multiple plaque characteristics measured by gadolinium-enhanced MRI among 1,700 participants in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI Study.
For the analyses presented here, 1,700 participants of the biracial ARIC Carotid MRI Study (~1,000 participants with thick carotid artery walls and ~700 randomly sampled participants) were evaluated for associations of MMP genetic variation with multiple plaque characteristics, including carotid artery wall thickness, lipid core and fibrous cap measures. MRI studies were performed on a 1.5T scanner equipped with a bilateral 4-element phased array carotid coil.
Fifty-one percent of the participants were female, 77% white, 23% African American, and the mean age was 70 years. MMP2 C-1306T variant genotypes (CT+TT) were significantly associated with higher cap thickness measures, but not with wall thickness or lipid core measures. Individuals with the CC genotype had approximately 0.1 mm thinner cap thickness compared to those carrying a T allele (p=0.02).
Genetic variation within the MMP2 promoter region was associated with cap thickness and therefore may influence the role of MMP2 in plaque vulnerability.
Atherosclerosis; Carotid MRI; Vulnerable Plaque; MMP; Genetics
We report a novel activatable NIR fluorescent probe for in vivo detection of cancer-related matrix metalloproteinase (MMP) activity. The probe is based on a triple-helical peptide substrate (THP) with high specificity for MMP-2 and MMP-9 relative to other members of the MMP family. MMP-2 and MMP-9 (also known as gelatinases) are specifically associated with cancer cell invasion and cancer-related angiogenesis. At the center of each 5 kDa peptide strand is a gelatinase sensitive sequence flanked by 2 Lys residues conjugated with NIR fluorescent dyes. Upon self-assembly of the triple-helical structure, the 3 peptide chains intertwine, bringing the fluorophores into close proximity and reducing fluorescence via quenching. Upon enzymatic cleavage of the triple-helical peptide, 6 labeled peptide chains are released, resulting in an amplified fluorescent signal. The fluorescence yield of the probe increases 3.8-fold upon activation. Kinetic analysis showed a rate of LS276-THP hydrolysis by MMP-2 (kcat/KM = 30,000 s−1M−1) similar to that of MMP-2 catalysis of an analogous fluorogenic THP. Administration of LS276-THP to mice bearing a human fibrosarcoma xenografted tumor resulted in a tumor fluorescence signal more than 5-fold greater than muscle. This signal enhancement was reduced by treatment with the MMP inhibitor Ilomostat, indicating that the observed tumor fluorescence was indeed enzyme mediated. These results are the first to demonstrate that triple-helical peptides are suitable for highly specific in vivo detection of tumor-related MMP-2 and MMP-9 activity.
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.
Although nitroglycerin (NTG) is effective for the acute relief in coronary ischemic diseases, its long-term benefits in mortality and morbidity have been questioned. The possibility has been raised that NTG may increase the activity of matrix metalloproteinases (MMP), which could lead to disruption and dislodging of atherosclerotic plaques. This study examined the broad effects of acute NTG exposure on the expression and activity of genes encoding MMP-9, as well as an array of ECM and adhesion molecules in THP-1 human macrophages. Gene array studies identified that while NTG exposure (100 µM, 48 hours) did not significantly increase MMP-9 gene expression, genes encoding testican-1, integrin α-1, thrombospondin-3, fibronectin-1 and MMP-26 were significantly down-regulated. On the other hand, genes encoding catenin β-1 and vascular cell adhesion molecule-1 were up-regulated. Real-time PCR studies confirmed significant down-regulation of testican-1 gene expression, but its protein expression was not significantly altered. NTG exposure, caused a significant increase in total MMP-9 protein expression (1.96-fold) and active MMP-9 (3.7-fold) concentrations. Recombinant MMP-9 was significantly activated by NTG and its dinitrate metabolites, indicating post-translation modification of this protein by organic nitrates. These results indicate that NTG exposure could broadly affect the gene expression and activity of proteases that govern the ECM cascade, thereby potentially altering atherosclerotic plaque stability.
Nitroglycerin; Matrix Metalloproteinase-9; Testican-1; Microarrays; Extracellular Matrix Proteases and Cell Adhesion Molecules
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.
Recently, the molecular mechanism responsible for the instability of atherosclerotic plaques has gradually become a hot topic among researchers and clinicians. Matrix metalloproteinases (MMPs) and vascular endothelial growth factor (VEGF) play an important role in the processes of formation and development of atherosclerosis. In this study, we established and employed the transwell co-culture system of rabbit aortic endothelial cells and smooth muscle cells to explore the relationship between fibrin (Fb), fibrinogen (Fg), and/or their degradation products (FDPs) in relation to the instability of atherosclerotic plaques; meanwhile, we observed the effects of Fg, Fb, and FDPs on the mRNA levels of MMPs and VEGF as well as on the activation of nuclear factor-kappa B (NF-κB). We concluded that Fb, Fg, and FDPs are involved in the progression of the instability of atherosclerotic plaques via increasing the expression of MMPs and VEGF. This effect might be mediated by the NF-кB pathway.
Fibrin(ogen); Fibrinogen degradation products; Atherosclerosis; Matrix metalloproteinase; Vascular endothelial growth factor; Nuclear factor-kappa B