Studying DNA G‑Quadruplex Aptamer by ¹⁹F NMR

In this study, we demonstrated that ¹⁹F NMR can be used to study the thrombin-binding aptamer (TBA) DNA G-quadruplex, widely used as a model structure for studying G-quadruplex aptamers. We systematically examined the structural feature of the TBA G-quadruplex aptamer with fluorine-19 (¹⁹F) labels at all of the thymidine positions. We successfully observed the structural change between the G-quadruplex and the unstructured single strand by ¹⁹F NMR spectroscopy. The thermodynamic parameters of these DNA G-quadruplex aptamers were also determined from the ¹⁹F NMR signals. We further showed that the ¹⁹F NMR method can be used to observe the complex formed by TBA G-quadruplex and thrombin. Our results suggest that ¹⁹F NMR spectroscopy is a useful approach to study the aptamer G-quadruplex structure

Impact of age on the region-specific concentrations of cytokine/chemokine in aortic tissue.

<p>Tissue concentrations of IL-6 (<b>A</b>), IL-10 (<b>B</b>), and RANTES (<b>C</b>) in the aortic arch (a), descending aorta (b), and abdominal aorta (c) of 16-, 32-, and 52-week-old apo E^−/− mice (red) and wild-type mice (yellow). The graphs present mean values of 3–4 samples. *p<0.05 vs. the respective region in 52-week-old wild-type mice; ^†p<0.05 vs. aortic arch, and <b>^#</b>p<0.05 vs. the descending aorta in apo E^−/− mice at 52 weeks.</p

Impact of age on the structure of the aorta in wild-type and apo E^−/− mice.

<p>(<b>A–C</b>) Atheromatous plaque area (<b>A</b>), elastin area (<b>B</b>), and collagen deposition (<b>C</b>) at each age and aorta region in apo E^−/− and wild-type mice. (<b>D–F</b>) Representative images of hematoxylin–eosin (<b>D</b>), Victoria blue (<b>E</b>), and picrosirius red (<b>F</b>) staining in the abdominal aorta of apo E^–/– mice at 52 weeks. Data are expressed as mean ± SEM [16 weeks: wild-type (n = 9), apo E^–/– mice (n = 5–6); 32 weeks: wild-type (n = 5–7), apo E^–/– mice (n = 6–7); 52 weeks: wild-type (n = 10), apo E^–/–- mice (n = 9–11)]. **p<0.01, ***p<0.001 vs. wild-type mice. Scale bar, 100 µm.</p

Impact of hyperlipidemia on the characteristics of the mice.

<p>Data are expressed as mean ± SEM (Parentheses indicate the number examined).</p><p>*P<0.05, **P<0.001, ***P<0.0001 vs. wild-type.</p><p>BW, body weight; SBP, systolic blood pressure; HR, heart rate; T-Cho, total cholesterol.</p><p>Apo E^−/−, apo E knockout mice.</p

Localization of IL-6 and RANTES in the abdominal aorta of 52-week old apo E^−/− mice.

<p>Representative immunostainings for IL-6 (<b>A</b>) and RANTES (<b>D</b>) in the abdominal aorta of 52-week-old apo E^−/− mice. Square areas were magnified to verify the co-localization of IL-6- (<b>B</b>) and F4/80-positive macrophages (<b>C</b>) or the co-localization of RANTES- (<b>E</b>) and CD3-positive T-lymphocytes (<b>F</b>) in serial sections. Scale bar, 100 µm (<b>A–D</b>) and 20 µm (<b>E, F</b>).</p

Relationship between lumen area and total vessel area in the abdominal aorta.

<p>(<b>A</b>) Impact of age on lumen and total vessel area in the abdominal aorta of apo E^−/− and wild-type mice. Data are expressed as mean ± SEM [16 weeks: wild-type (n = 9), apo E^−/− mice (n = 5); 32 weeks: wild-type (n = 5), apo E^−/− mice (n = 7); 52 weeks: wild-type (n = 10), apo E^−/− mice (n = 9)]. **p<0.01 vs. wild-type mice. (<b>B, C</b>) Relationship between lumen area and total vessel area (<b>B</b>) or plaque area (<b>C</b>) in 16-, 32-, and 52-week-old apo E^−/− mice (black square) and wild-type mice (white circle).</p

Relationship between plaque area and adventitial inflammation in the abdominal aorta.

<p>(<b>A–C</b>) Liner regression between plaque/media area and the number of [macrophages (<b>A</b>), T-lymphocytes (<b>B</b>), and microvessels (<b>C</b>)] in the adventitia of 52-week-old apo E^−/− mice.</p

Impact of age on adventitia inflammatory cells, microvessels, and perivascular adipocytes.

<p>(<b>A–C</b>) Numbers of F4/80-positive macrophages (<b>A</b>), CD3-positive T-lymphocytes (<b>B</b>), and CD31-positive microvessels (<b>C</b>) in the adventitia of the aorta at each age and region in apo E^−/− and wild-type mice. (<b>D–F</b>) Representative images of F4/80 (<b>D</b>), CD3 (<b>E</b>), and CD31 (<b>F</b>) immunostaining in the abdominal aorta of apo E^−/− mice at 52 weeks. Arrows indicate CD31-positive microvessels in the adventitia. (<b>G, H</b>) Representative hematoxylin–eosin staining of perivascular adipose tissue in the descending aorta (<b>G</b>) and abdominal aorta (<b>H</b>) of 52-week-old apo E^−/− mice. (<b>I</b>) Diameter of adipocytes surrounding the aortic arch, descending aorta, and abdominal aorta. Data are expressed as mean ± SEM [16 weeks: wild-type (n = 9), apo E^−/− mice (n = 5–6); 32 weeks: wild-type (n = 5–7), apo E^−/− mice (n = 6–7); 52 weeks: wild-type (n = 10), apo E^−/− mice (n = 9–11)]. *p<0.05, **p<0.01 vs. wild-type mice. Scale bar, 100 µm.</p

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