[18F]FDG Uptake in the Aortic Wall Smooth Muscle of Atherosclerotic Plaques in the Simian Atherosclerosis Model

Atherosclerosis is a self-sustaining inflammatory fibroproliferative disease that progresses in discrete stages and involves a number of cell types and effector molecules. Recently, [18F]fluoro-2-deoxy-D-glucose- ([18F]FDG-) positron emission tomography (PET) has been suggested as a tool to evaluate atherosclerotic plaques by detecting accumulated macrophages associated with inflammation progress. However, at the cellular level, it remains unknown whether only macrophages exhibit high uptake of [18F]FDG. To identify the cellular origin of [18F]FDG uptake in atherosclerotic plaques, we developed a simian atherosclerosis model and performed PET and ex vivo macro- and micro-autoradiography (ARG). Increased [18F]FDG uptake in the aortic wall was observed in high-cholesterol diet-treated monkeys and WHHL rabbits. Macro-ARG of [18F]FDG in aortic sections showed that [18F]FDG was accumulated in the media and intima in the simian model as similar to that in WHHL rabbits. Combined analysis of micro-ARG with immunohistochemistry in the simian atherosclerosis model revealed that most cellular [18F]FDG uptake observed in the media was derived not only from the infiltrated macrophages in atherosclerotic plaques but also from the smooth muscle cells (SMCs) of the aortic wall in atherosclerotic lesions

Successful serial imaging of the mouse cerebral arteries using conventional 3-T magnetic resonance imaging

Serial imaging studies can be useful in characterizing the pathologic and physiologic remodeling of cerebral arteries in various mouse models. We tested the feasibility of using a readily available, conventional 3-T magnetic resonance imaging (MRI) to serially image cerebrovascular remodeling in mice. We utilized a mouse model of intracranial aneurysm as a mouse model of the dynamic, pathologic remodeling of cerebral arteries. Aneurysms were induced by hypertension and a single elastase injection into the cerebrospinal fluid. For the mouse cerebrovascular imaging, we used a conventional 3-T MRI system and a 40-mm saddle coil. We used non-enhanced magnetic resonance angiography (MRA) to detect intracranial aneurysm formation and T2-weighted imaging to detect aneurysmal subarachnoid hemorrhage. A serial MRI was conducted every 2 to 3 days. MRI detection of aneurysm formation and subarachnoid hemorrhage was compared against the postmortem inspection of the brain that was perfused with dye. The imaging times for the MRA and T2-weighted imaging were 3.7±0.5 minutes and 4.8±0.0 minutes, respectively. All aneurysms and subarachnoid hemorrhages were correctly identified by two masked observers on MRI. This MRI-based serial imaging technique was useful in detecting intracranial aneurysm formation and subarachnoid hemorrhage in mice

Vascular Smooth Muscle Cells Stimulate Platelets and Facilitate Thrombus Formation through Platelet CLEC-2: Implications in Atherothrombosis

<div><p>The platelet receptor CLEC-2 is involved in thrombosis/hemostasis, but its ligand, podoplanin, is expressed only in advanced atherosclerotic lesions. We investigated CLEC-2 ligands in vessel walls. Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. VSMCs stimulated platelet granule release and supported thrombus formation under flow, dependent on CLEC-2. The time to occlusion in a FeCl₃-induced animal thrombosis model was significantly prolonged in the absence of CLEC-2. Because the internal elastic lamina was lacerated in our FeCl₃-induced model, we assume that the interaction between CLEC-2 and its ligands in VSMCs induces thrombus formation. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. However, S100A13 is not responsible for the above-described VSMC-induced platelet activation, because S100A13 is not expressed on the surface of normal VSMCs. S100A13 was released upon oxidative stress and expressed in the luminal area of atherosclerotic lesions. Suspended S100A13 did not activate platelets, but immobilized S100A13 significantly increased thrombus formation on collagen-coated surfaces. Taken together, we proposed that VSMCs stimulate platelets through CLEC-2, possibly leading to thrombus formation after plaque erosion and stent implantation, where VSMCs are exposed to blood flow. Furthermore, we identified S100A13 as one of the ligands on VSMCs.</p></div

Coronary artery smooth muscle cells (CASMCs) stimulated release of α and dense granule contents through CLEC-2.

<p>Washed platelets obtained from the CLEC-2-deficient chimeras were incubated with buffer, CASMCs, CRP, rhodocytin, or lysis buffer for 10 min. The platelets were centrifuged. The amount of secreted 5-hydroxytryptamine (5-HT) (A) or platelet-derived growth factor (PDGF) (B) in the supernatants was measured by ELISA. Platelet lysates were used to measure the total amount of 5-HT or PDGF stored in platelets. The results were expressed as the percentage of secreted 5-HT or PDGF relative to the total amount stored in platelets ± SE (A. n = 9 from three independent experiments, B. n = 12 from four independent experiments). Three asterisks denote p < 0.005.</p

S100A13 potentiated thrombus formation on collagen-coated surfaces under flow.

<p>(A) Anti-coagulated human whole blood was perfused into cell microscopy chambers coated with collagen or collagen plus S100A13 for 10 min at a shear rate of 1500 s⁻¹. Adherent platelets were visualized using a fluorescence video microscope. Movie data were converted into sequential photo images. (B) Thrombus volume was quantified. After 5 min of perfusion, adherent platelets were visualized by confocal laser microscopy, and the z-stack data were analyzed. The thrombus volume was expressed as the cIFI per image (404374 μm²). The graph illustrates the percentage of the surfaces coated with collagen only cIFI ± SE (n = 6, from two independent experiments).</p

Time to thrombotic occlusion of vessels stimulated by FeCl₃, but not by green light and dye, was significantly delayed in the CLEC-2-deficient chimeras.

<p>The femoral artery of anesthetized wild type (WT) chimeras or CLEC-2-deficient chimeras was exposed by blunt dissection. A) Time to vessel occlusion in the FeCl₃ model: a 1strip of filter paper saturated with 10% FeCl₃ was applied to the adventitial surface of the exposed femoral artery. B) Time to occlusion in the photochemically induced thrombosis (PIT) model. After 5 min of irradiation to the exposed femoral artery, rose Bengal was infused. The time to thrombotic vessel occlusion of the artery was measured by monitoring femoral blood flow using a Doppler blood flow velocimeter. Each symbol represents one individual. The mean time to occlusion ± SE is indicated in the graphs. Two asterisks denote p < 0.01.</p

S100A13 is not expressed on the surface of coronary artery smooth muscle cells (CASMCs), but oxidative stress induced surface expression of S100A13.

<p>A) Binding of recombinant S100A13 on the surface of CASMCs were examined by flow cytometory. CASMCs were preincubated with vehicle only (1st panel) or recombinant S100A13 in the presence of vehicle (2^nd panel), 0.1 mM CaCl₂ (3^rd panel), 1 mM EDTA (4^th panel), or 0.1 mM CaCl₂ + 1 mM EDTA (5^th panel). After excess of the recombinant protein was removed by centrifugation, cells were incubated with control mouse IgG (filled) or anti-S100A13 antibody (line), followed by Alexa Flour 488-conjugated anti-mouse IgG. B) Surface expression of endogenous S100A13 was analyzed by flow cytometry. CASMCs pretreated with indicated concentrations of H₂O₂ were incubated with control mouse IgG (filled) or anti-S100A13 antibody (line), followed by Alexa Flour 488-conjugated anti-mouse IgG.</p

Recombinant S100A13 protein associated with CLEC-2.

<p>A) Different concentrations of His-S100A13 were flowed over an immobilized hCLEC-2-rFc2 or a control rFc2-coated surface. The arrows indicate the beginning and the end of perfusion of S100A13. The results from one experiment are shown that is representative of the other three. RU indicates resonance units. B) (i) Platelets spreading on the surface of BSA, collagen, or S100A13 were investigated. (ii) Magnified images of adhered WT platelets on the surfaces coated with BSA or recombinant S100A13. (iii) Quantification of adherent platelets in the images in (i). Adherent platelets were counted. C) Western blotting with anti-S100A13 or anti-β3 integrin antibody. Plt represents platelets. The data are representative of three experiments.</p