Molecular imaging of MMP activity discriminates unstable from stable plaque phenotypes in shear-stress induced murine atherosclerosis.

PURPOSE: As atherosclerotic plaque ruptures are the primary cause of ischaemic events, their preventive identification by imaging remains a clinical challenge. Matrix metalloproteinases (MMP) are involved in plaque progression and destabilisation and are therefore promising targets to characterize rupture-prone unstable plaques. This study aims at evaluating MMP imaging to discriminate unstable from stable plaque phenotypes. METHODS: ApoE deficient mice (ApoE-/-) on a high cholesterol diet underwent implantation of a tapered cuff around the right common carotid artery (CCA) inducing a highly inflamed atherosclerotic plaque upstream (US) and a more stable plaque phenotype downstream (DS) of the cuff. 8 weeks after surgery, the MMP inhibitor-based photoprobe Cy5.5-AF443 was administered i.v. 3h prior to in situ and ex vivo fluorescence reflectance imaging of the CCAs. Thereafter, CCAs were analysed regarding plaque size, presence of macrophages, and MMP-2 and MMP-9 concentrations by immunohistochemistry and ELISA. RESULTS: We found a significantly higher uptake of Cy5.5-AF443 in US as compared to DS plaques in situ (1.29 vs. 1.06 plaque-to-background ratio; p<0.001), which was confirmed by ex vivo measurements. Immunohistochemistry revealed a higher presence of macrophages, MMP-2 and MMP-9 in US compared to DS plaques. Accordingly, MMP-2 concentrations were significantly higher in US plaques (47.2±7.6 vs. 29.6±4.6 ng/mg; p<0.05). CONCLUSIONS: In the ApoE-/- cuff model MMP-2 and MMP-9 activities are significantly higher in upstream low shear stress-induced unstable atherosclerotic plaques as compared to downstream more stable plaque phenotypes. MMP inhibitor-based fluorescence molecular imaging allows visualization of these differences in shear stress-induced atherosclerosis.

Imaging, myeloid precursor immortalization, and genome editing for defining mechanisms of leukocyte recruitment in vivo.

Recruitment of leukocytes from the blood to sites of inflammation poses a promising target for new diagnostic and therapeutic approaches. We aimed to develop a novel method to non-invasively analyze molecular mechanisms of leukocyte migration in pre-clinical models of inflammation in vivo. Methods: We used the ER-HoxB8 system to transiently immortalize murine myeloid precursors from wildtype and CD18- as well as MRP14-deficient mice. A VLA4α-/- cell line was generated by CRISPR/Cas9-mediated gene editing. We analyzed the migration of wildtype and knockout leukocytes in vivo by optical and nuclear imaging in mice with irritant contact dermatitis, cutaneous granuloma, experimental arthritis and myocardial infarction. Results: Transient immortalization, gene editing and in vivo imaging can be combined to analyze migratory mechanisms of murine leukocytes, even for gene deletions resulting in lethal phenotypes in mice. We reliably confirmed known migratory defects of leukocytes deficient for the adhesion molecules CD18 or VLA4α. Also, using our new method we identified a new role of the most abundant calcium-binding proteins in phagocytes and major alarmins in many inflammatory diseases, MRP8 and MRP14, for transmigration in vivo. Conclusion: We provide a combinatorial approach to rapidly characterize molecular mechanisms of leukocyte recruitment in vivo, with the potential to aid in identification of diagnostic and therapeutic targets in inflammatory pathologies.

Effect of a multinutrient intervention after ischemic stroke in female C57Bl/6 mice.

Stroke can affect females very differently from males, and therefore preclinical research on underlying mechanisms and the effects of interventions should not be restricted to male subjects, and treatment strategies for stroke should be tailored to benefit both sexes. Previously, we demonstrated that a multinutrient intervention (Fortasyn) improved impairments after ischemic stroke induction in male C57Bl/6 mice, but the therapeutic potential of this dietary treatment remained to be investigated in females. We now induced a transient middle cerebral artery occlusion (tMCAo) in C57Bl/6 female mice and immediately after surgery switched to either Fortasyn or an isocaloric Control diet. The stroke females performed several behavioral and motor tasks before and after tMCAo and were scanned in an 11.7 Tesla magnetic resonance imaging (MRI) scanner to assess brain perfusion, integrity, and functional connectivity. To assess brain plasticity, inflammation, and vascular integrity, immunohistochemistry was performed after killing of the mice. We found that the multinutrient intervention had diverse effects on the stroke-induced impairments in females. Similar to previous observations in male stroke mice, brain integrity, sensorimotor integration and neurogenesis benefitted from Fortasyn, but impairments in activity and motor skills were not improved in female stroke mice. Overall, Fortasyn effects in the female stroke mice seem more modest in comparison to previously investigated male stroke mice. We suggest that with further optimization of treatment protocols more information on the efficacy of specific interventions in stroked females can be gathered. This in turn will help with the development of (gender-specific) treatment regimens for cerebrovascular diseases such as stroke. This article is part of the Special Issue "Vascular Dementia".

A specific dietary intervention to restore brain structure and function after ischemic stroke.

Occlusion of the middle cerebral artery (MCAo) is among the most common causes of ischemic stroke in humans. Cerebral ischemia leads to brain lesions existing of an irreversibly injured core and an ischemic boundary zone, the penumbra, containing damaged but potentially salvageable tissue. Using a transient occlusion (30 min) of the middle cerebral artery (tMCAo) mouse model in this cross-institutional study we investigated the neurorestorative efficacy of a dietary approach (Fortasyn) comprising docosahexaenoic acid, eicosapentaenoic acid, uridine, choline, phospholipids, folic acid, vitamins B12, B6, C, and E, and selenium as therapeutic approach to counteract neuroinflammation and impairments of cerebral (structural+functional) connectivity, cerebral blood flow (CBF), and motor function. Male adult C57BL/6j mice were subjected to right tMCAo using the intraluminal filament model. Following tMCAo, animals were either maintained on Control diet or switched to the multicomponent Fortasyn diet. At several time points after tMCAo, behavioral tests, and MRI and PET scanning were conducted to identify the impact of the multicomponent diet on the elicited neuroinflammatory response, loss of cerebral connectivity, and the resulting impairment of motor function after experimental stroke. Mice on the multicomponent diet showed decreased neuroinflammation, improved functional and structural connectivity, beneficial effect on CBF, and also improved motor function after tMCAo. Our present data show that this specific dietary intervention may have beneficial effects on structural and functional recovery and therefore therapeutic potential after ischemic stroke.

Imaging Reveals the Connection Between Spontaneous Coronary Plaque Ruptures, Atherothrombosis, and Myocardial Infarctions in HypoE/SRBI-/- Mice.

UNLABELLED: The hyperlipidemic mouse model HypoE/SRBI(-/-) has been shown to develop occlusive coronary atherosclerosis followed by myocardial infarctions and premature deaths in response to high-fat, high-cholesterol diet (HFC). However, the causal connection between myocardial infarctions and atherosclerotic plaque rupture events in the coronary arteries has not been investigated so far. The objective of this study was to assess whether diet-induced coronary plaque ruptures trigger atherothrombotic occlusions, resulting in myocardial infarctions in HFC-fed HypoE/SRBI(-/-) mice. METHODS: HypoE/SRBI(-/-) mice were characterized with respect to the individual dynamics of myocardial infarctions and features of infarct-related coronary atherosclerosis by serial noninvasive molecular and functional imaging, histopathology, and a pharmaceutical intervention. Detailed histologic analysis of whole mouse hearts was performed when spontaneously occurring acute myocardial infarctions were diagnosed by imaging. RESULTS: Using the imaging-triggered approach, we discovered thrombi in 32 (10.8%) of all 296 atherosclerotic coronary plaques in 14 HFC-fed HypoE/SRBI(-/-) mice. These thrombi typically were found in arteries presenting with inflammatory plaque phenotypes. Acetylsalicylic acid treatment did not attenuate the development of atherosclerotic coronary plaques but profoundly reduced the incidence of premature deaths, the number of thrombi (7 in 249 plaques), and also the degree of inflammation in the culprit lesions. CONCLUSION: HFC-induced ruptures of coronary plaques trigger atherothrombosis, vessel occlusions, myocardial infarctions, and sudden death in these mice. Thus, the HypoE/SRBI(-/-) mouse model mimics major features of human coronary heart disease and might therefore be a valuable model for the investigation of molecular and cellular parameters driving plaque rupture-related events and the development of new interventional approaches.

Generation of functional endothelial-like cells from adult mouse germline-derived pluripotent stem cells.

Functional endothelial cells and their progenitors are required for vascular development, adequate vascular function, vascular repair and for cell-based therapies of ischemic diseases. Currently, cell therapy is limited by the low abundance of patient-derived cells and by the functional impairment of autologous endothelial progenitor cells (EPCs). In the present study, murine germline-derived pluripotent stem (gPS) cells were evaluated as a potential source for functional endothelial-like cells. Cells displaying an endothelial cell-like morphology were obtained from gPS cell-derived embryoid bodies using a combination of fluorescence-activated cell sorting (FACS)-based selection of CD31-positive cells and their subsequent cultivation on OP9 stromal cells in the presence of VEGF-A. Real-time reverse transcriptase polymerase chain reaction, FACS analysis and immunofluorescence staining showed that the gPS cell-derived endothelial-like cells (gPS-ECs) expressed endothelial cell-specific markers including von Willebrand Factor, Tie2, VEGFR2/Flk1, intercellular adhesion molecule 2 and vascular endothelial-cadherin. The high expression of ephrin B2, as compared to Eph B4 and VEGFR3, suggests an arterial rather than a venous or lymphatic differentiation. Their capability to take up Dil-conjugated acetylated low-density lipoprotein and to form capillary-like networks on matrigel confirmed their functionality. We conclude that gPS cells could be a novel source of endothelial cells potentially suitable for regenerative cell-based therapies for ischemic diseases.