Cytokine Response to Diet and Exercise Affects Atheromatous Matrix Metalloproteinase-2/9 Activity in Mice.

BACKGROUND: The aim of this study is to identify the principal circulating factors that modulate atheromatous matrix metalloproteinase (MMP) activity in response to diet and exercise.Methods and Results:Apolipoprotein-E knock-out (ApoE-/-) mice (n=56) with pre-existing plaque, fed either a Western diet (WD) or normal diet (ND), underwent either 10 weeks of treadmill exercise or had no treatment. Atheromatous MMP activity was visualized using molecular imaging with a MMP-2/9 activatable near-infrared fluorescent (NIRF) probe. Exercise did not significantly reduce body weight, visceral fat, and plaque size in either WD-fed animals or ND-fed animals. However, atheromatous MMP-activity was different; ND animals that did or did not exercise had similarly low MMP activities, WD animals that did not exercise had high MMP activity, and WD animals that did exercise had reduced levels of MMP activity, close to the levels of ND animals. Factor analysis and path analysis showed that soluble vascular cell adhesion molecule (sVCAM)-1 was directly positively correlated to atheromatous MMP activity. Adiponectin was indirectly negatively related to atheromatous MMP activity by way of sVCAM-1. Resistin was indirectly positively related to atheromatous MMP activity by way of sVCAM-1. Visceral fat amount was indirectly positively associated with atheromatous MMP activity, by way of adiponectin reduction and resistin elevation. MMP-2/9 imaging of additional mice (n=18) supported the diet/exercise-related anti-atherosclerotic roles for sVCAM-1. CONCLUSIONS: Diet and exercise affect atheromatous MMP activity by modulating the systemic inflammatory milieu, with sVCAM-1, resistin, and adiponectin closely interacting with each other and with visceral fat.

Photodynamic therapy using a protease-mediated theranostic agent reduces cathepsin-B activity in mouse atheromata in vivo.

OBJECTIVE: To investigate whether an intravenously injected cathepsin-B activatable theranostic agent (L-SR15) would be cleaved in and release a fluorescent agent (chlorin-e6) in mouse atheromata, allowing both the diagnostic visualization and therapeutic application of these fluorophores as photosensitizers during photodynamic therapy to attenuate plaque-destabilizing cathepsin-B activity by selectively eliminating macrophages. APPROACH AND RESULTS: Thirty-week-old apolipoprotein E knock-out mice (n=15) received intravenous injection of L-SR15 theranostic agent, control agent D-SR16, or saline 3× (D0, D7, D14). Twenty-four hours after each injection, the bilateral carotid arteries were exposed, and Cy5.5 near-infrared fluorescent imaging was performed. Fluorescent signal progressively accumulated in the atheromata of the L-SR15 group animals only, indicating that photosensitizers had been released from the theranostic agent and were accumulating in the plaque. After each imaging session, photodynamic therapy was applied with a continuous-wave diode-laser. Additional near-infrared fluorescent imaging at a longer wavelength (Cy7) with a cathepsin-B-sensing activatable molecular imaging agent showed attenuation of cathepsin-B-related signal in the L-SR15 group. Histological studies demonstrated that L-SR15-based photodynamic therapy decreased macrophage infiltration by inducing apoptosis without significantly affecting plaque size or smooth muscle cell numbers. Toxicity studies (n=24) showed that marked erythematous skin lesion was generated in C57/BL6 mice at 24 hours after intravenous injection of free chlorin-e6 and ultraviolet light irradiation; however, L-SR15 or saline did not cause cutaneous phototoxicity beyond that expected of ultraviolet irradiation alone, neither did we observe systemic toxicity or neurobehavioral changes. CONCLUSIONS: This is the first study showing that macrophage-secreted cathepsin-B activity in atheromata could be attenuated by photodynamic therapy using a protease-mediated theranostic agent.

Protease imaging of human atheromata captures molecular information of atherosclerosis, complementing anatomic imaging.

OBJECTIVE: There is hope that molecular imaging can identify vulnerable atherosclerotic plaques. However, there is a paucity of clinical translational data to guide the future development of this field. Here, we cross-correlate cathepsin-B or matrix metalloproteinase-2/-9 molecular optical imaging data of human atheromata or emboli with conventional imaging data, clinical data, and histopathologic data. METHODS AND RESULTS: Fifty-two patients undergoing carotid endarterectomy (41 atheromata) or carotid stenting (15 captured emboli) were studied with protease-activatable imaging probes. We show that protease-related fluorescent signal in carotid atheromata or in emboli closely reflects the pathophysiologic alterations of plaque inflammation and statin-mediated therapeutic effects on plaque inflammation. Inflammation-related fluorescent signal was observed in the carotid bifurcation area and around ulcero-hemorrhagic lesions. Pathologically proven unstable plaques had high cathepsin-B-related fluorescent signal. The distribution patterns of the mean cathepsin-B imaging signals showed a difference between the symptomatic vs asymptomatic plaque groups. However, the degree of carotid stenosis or ultrasonographic echodensity was weakly correlated with the inflammatory proteolytic enzyme-related signal, suggesting that molecular imaging yields complimentary new information not available to conventional imaging. CONCLUSIONS: These results could justify and facilitate clinical trials to evaluate the use of protease-sensing molecular optical imaging in human atherosclerosis patients.

Atorvastatin and clopidogrel interfere with photosensitization in vitro.

Photodynamic therapy (PDT) has been used to eliminate undesired cells by using a combination of photosensitizers and light illumination to generate reactive oxygen species. There is great interest in applying PDT to atherosclerosis; preferential destruction of pro-inflammatory macrophages in atheromata might attenuate plaque growth or rupture-prone vulnerability. Here, we report on a previously unknown interaction between cardiovascular drugs that are commonly prescribed for atherosclerosis patients and the cytolytic effects of photodynamic therapy using Cathepsin B activatable photosensitizer L-SR15 on murine macrophage Raw 264.7 cells in culture. Atorvastatin and clopidogrel significantly interfered with in vitro photosensitization effect while aspirin did this to a lesser extent; these drugs did not change the efficiency of cellular uptake of L-SR15 after in vitro photosensitization. A photosensitization interference effect of atorvastatin and clopidogrel was also observed when using a conventional photosensitizer free Ce6 or NCI-H1299 cancer cells. Considering the clinical implications of PDT, our study merits further investigation in clinical settings as well as in animal models.

Molecular imaging of cathepsin B proteolytic enzyme activity reflects the inflammatory component of atherosclerotic pathology and can quantitatively demonstrate the antiatherosclerotic therapeutic effects of atorvastatin and glucosamine.

Inflammation in atherosclerotic plaques causes plaque vulnerability and rupture, leading to thromboembolic complications. Cathepsin B (CatB) proteases secreted by macrophages play a major role in plaque inflammation. We used a CatB-activatable near-infrared fluorescence (NIRF) imaging agent to demonstrate the inflammatory component in mice atheromata and the atherosclerosis- modulating effects of atorvastatin or glucosamine treatments. Apolipoprotein E knockout mice (n = 35) were fed normal chow, a Western diet, a Western diet + atorvastatin, a Western diet + glucosamine, or a Western diet + atorvastatin + glucosamine for 14 weeks. Twenty-four hours after the intravenous injection of a CatB-activatable probe, ex vivo NIRF imaging of the aortas and brains was performed, followed by histology. The CatB-related signal, observed in the aortas but not in the cerebral arteries, correlated very well with protease activity and the presence of macrophages on histology. Animals on Western diets could be distinguished from animals on a normal diet. The antiatherosclerotic effects of atorvastatin and glucosamine could be demonstrated, with reduced CatB-related signal compared with untreated animals. Plaque populations were heterogeneous within individuals, with some plaques showing a high and others a lower CatB-related signal. These differences in signal intensity could not be predicted by visual inspection of the plaques but did correlate with histologic evidence of inflammation in every case. This suggests that vulnerable inflamed plaques can be identified by optical molecular imaging.

Characterization of partial ligation-induced carotid atherosclerosis model using dual-modality molecular imaging in ApoE knock-out mice.

BACKGROUND: Recently, partial ligation of the common carotid artery (CCA) was reported to induce carotid atheromata rapidly in apolipoprotein-E knockout (ApoE(-/-)) mice. We investigated this new atherosclerosis model by using combined matrix-metalloproteinase (MMP) near-infrared fluorescent (NIRF) imaging and macrophage-tracking luciferase imaging. METHODOLOGY AND PRINCIPAL FINDINGS: Partial ligation of the left CCA was performed in 10-week-old ApoE(-/-) mice on a high fat diet (n=33); the internal and external carotid arteries and occipital artery were ligated, while the superior thyroid artery was left intact. Two thirds of the animals were treated with either LiCl or atorvastatin. At 1-week, Raw264.7 macrophages modified to express the enhanced firefly-luciferase reporter gene (10(7) Raw-luc cells) were injected intravenously. At 2-week, NIRF molecular imaging visualized strong MMP-2/9 activity in the ligated area of the left CCA as well as in the aortic arch. Left-to-right ratios of the NIRF signal intensities in the CCA had a decreasing gradient from the highest value in the upper-most ligated area to the lowest value in the lower-most region adjacent to the aortic arch. Luciferase imaging showed that most Raw-luc macrophages were recruited to the ligated area of the CCA rather than to the aortic arch, despite similarly strong MMP-2/9-related NIRF signal intensities in both areas. In addition, LiCl or atorvastatin could reduce MMP-2/9 activity in the aortic arch but not in the ligated area of the CCA. CONCLUSIONS/SIGNIFICANCE: This is the first molecular imaging study to characterize the partial ligation-induced carotid atherosclerosis model. Molecularly divergent types of atherosclerosis were identified: conventional lipogenic atherosclerosis in the aorta vs. flow-related mechanical atherosclerosis in the partially ligated left system.

Exercise attenuates matrix metalloproteinase activity in preexisting atherosclerotic plaque.

OBJECTIVE: Few studies have investigated if exercise by itself has anti-atherosclerotic effects, without combining interventions with a low-fat diet. We studied the effects of exercise as a stand-alone intervention on preexisting atheromata by measuring not only plaque size but also the levels of plaque-destabilizing matrix-metalloproteinase (MMP) activity in vivo. METHODS AND RESULTS: We used near-infrared fluorescent (NIRF) molecular imaging with an MMP-2/9 activatable NIRF probe to visualize the inflammatory protease activity within preexisting atheromata of 17-week-old ApoE(-/-) mice on: (a) normal chow diet (NCD), (b) Western diet (WD), and (c) WD with treadmill exercise for 10 weeks. We also measured tissue levels of aortic lipid peroxidation (LPO) and plasma levels of glucose/lipid/cytokine profiles. Exercise did not attenuate growth of preexisting atheromatous plaques. However, exercise strongly decreased proteolytic activity in plaques for animals on WD, with levels decreasing almost to NCD levels. Exercise was associated with decreased aortic LPO levels and increased blood adiponectin/leptin levels; however, exercise did not affect WD-consumption/weight-gain or improve blood glucose/lipid profiles. CONCLUSIONS: Exercise training reduced aortic MMP activity in mice with preexisting atheromata, even though they remained on a high fat diet and plaque-growth was not attenuated.

Early treadmill training promotes motor function after hemorrhagic stroke in rats.

Rehabilitation after a stroke is very important because it has beneficial effects on brain function, including the promotion of plasticity. However, an optimal time window for rehabilitation interventions after hemorrhagic stroke has not been clearly defined. The aim of this study was to determine whether early exercise training initiated 24h after an intracerebral hemorrhage (ICH) might enhance neurologic recovery more than exercise initiated 1 week after ICH without hematoma expansion and edema volume increase. We subjected adult male Sprague-Dawley rats to experimental ICH by the intrastriatal administration of bacterial collagenase. The rats were randomly divided into the following 2 groups: early training group (treadmill exercise started 24h post-ICH; n=18) and late training group (treadmill exercise started 1-week post-ICH; n=18). Two weeks after surgery we performed neurologic tests (rota-rod, modified limb-placing, and adhesive-dot removal tests), and measured hematoma volumes and brain water content. In the late training group, compared with the pre-ICH performance on the rota-rod test (98.3+/-69.4s), the animals had significantly worse performance after the post-ICH rehabilitation (40.5+/-52.6s; p<0.01, paired t-test). In the early training group however, the motor performance after the post-ICH rehabilitation (56.4+/-73.5s) was not significantly different from the baseline pre-ICH performance (79.8+/-33.9s; p=0.24). There were no significant differences between the two groups with respect to the other neurologic tests. Early exercise did not increase hematoma size or brain water content. Early treadmill training could be performed safely, and enhanced motor recovery in a rat model of ICH. Further studies are required to translate the results into clinical significance.