Activated Platelets in Carotid Artery Thrombosis in Mice Can Be Selectively Targeted with a Radiolabeled Single-Chain Antibody

BACKGROUND: Activated platelets can be found on the surface of inflamed, rupture-prone and ruptured plaques as well as in intravascular thrombosis. They are key players in thrombosis and atherosclerosis. In this study we describe the construction of a radiolabeled single-chain antibody targeting the LIBS-epitope of activated platelets to selectively depict platelet activation and wall-adherent non-occlusive thrombosis in a mouse model with nuclear imaging using in vitro and ex vivo autoradiography as well as small animal SPECT-CT for in vivo analysis. METHODOLOGY/PRINCIPAL FINDINGS: LIBS as well as an unspecific control single-chain antibody were labeled with (111)Indium ((111)In) via bifunctional DTPA ( = (111)In-LIBS/(111)In-control). Autoradiography after incubation with (111)In-LIBS on activated platelets in vitro (mean 3866 ± 28 DLU/mm(2), 4010 ± 630 DLU/mm(2) and 4520 ± 293 DLU/mm(2)) produced a significantly higher ligand uptake compared to (111)In-control (2101 ± 76 DLU/mm(2), 1181 ± 96 DLU/mm(2) and 1866 ± 246 DLU/mm(2)) indicating a specific binding to activated platelets; P<0.05. Applying these findings to an ex vivo mouse model of carotid artery thrombosis revealed a significant increase in ligand uptake after injection of (111)In-LIBS in the presence of small thrombi compared to the non-injured side, as confirmed by histology (49630 ± 10650 DLU/mm(2) vs. 17390 ± 7470 DLU/mm(2); P<0.05). These findings could also be reproduced in vivo. SPECT-CT analysis of the injured carotid artery with (111)In-LIBS resulted in a significant increase of the target-to-background ratio compared to (111)In-control (1.99 ± 0.36 vs. 1.1 ± 0.24; P < 0.01). CONCLUSIONS/SIGNIFICANCE: Nuclear imaging with (111)In-LIBS allows the detection of platelet activation in vitro and ex vivo with high sensitivity. Using SPECT-CT, wall-adherent activated platelets in carotid arteries could be depicted in vivo. These results encourage further studies elucidating the role of activated platelets in plaque pathology and atherosclerosis and might be of interest for further developments towards clinical application

Cannabinoid Receptor 2 Signaling Does Not Modulate Atherogenesis in Mice

BACKGROUND:Strong evidence supports a protective role of the cannabinoid receptor 2 (CB(2)) in inflammation and atherosclerosis. However, direct proof of its involvement in lesion formation is lacking. Therefore, the present study aimed to characterize the role of the CB(2) receptor in Murine atherogenesis. METHODS AND FINDINGS:Low density lipoprotein receptor-deficient (LDLR(-/-)) mice subjected to intraperitoneal injections of the selective CB(2) receptor agonist JWH-133 or vehicle three times per week consumed high cholesterol diet (HCD) for 16 weeks. Surprisingly, intimal lesion size did not differ between both groups in sections of the aortic roots and arches, suggesting that CB(2) activation does not modulate atherogenesis in vivo. Plaque content of lipids, macrophages, smooth muscle cells, T cells, and collagen were also similar between both groups. Moreover, CB(2) (-/-)/LDLR(-/-) mice developed lesions of similar size containing more macrophages and lipids but similar amounts of smooth muscle cells and collagen fibers compared with CB(2) (+/+)/LDLR(-/-) controls. While JWH-133 treatment reduced intraperitoneal macrophage accumulation in thioglycollate-elicited peritonitis, neither genetic deficiency nor pharmacologic activation of the CB(2) receptor altered inflammatory cytokine expression in vivo or inflammatory cell adhesion in the flow chamber in vitro. CONCLUSION:Our study demonstrates that both activation and deletion of the CB(2) receptor do not relevantly modulate atherogenesis in mice. Our data do not challenge the multiple reports involving CB(2) in other inflammatory processes. However, in the context of atherosclerosis, CB(2) does not appear to be a suitable therapeutic target for reduction of the atherosclerotic plaque

Guanosine 5'-[beta-thio]triphosphate selectively activates calcium signaling in mast cells.

In rat peritoneal mast cells, the activation of GTP-binding proteins (G proteins) by guanosine 5'-[gamma-thio]triphosphate GTP[gamma S] has been found to induce a transient rise in intracellular calcium as well as degranulation. A G protein that couples to phospholipase C (Gp) is thought to mediate the calcium response, whereas degranulation is mediated by a different G protein, termed Ge. In an attempt to activate mast-cell G proteins more selectively, the GTP analogues guanosine 5'-[alpha-thio]triphosphate (GTP[alpha S]) and guanosine 5'-[beta-thio]triphosphate (GTP[beta S]) (RP and SP diastereomers) were introduced into mast cells by means of patch pipettes. Degranulation and free intracellular calcium were monitored by cell capacitance and fura-2 measurements, respectively. It was found that RP-GTP[alpha S], like GTP[gamma S], induced both calcium release and exocytosis. In contrast, RP-GTP[beta S] induced repetitive calcium spikes that were not regularly accompanied by exocytosis. These results suggest that RP-GTP[beta S] selectively activates calcium signaling in mast cells. The RP-GTP[beta S]-induced oscillations were independent of extracellular calcium. They were absent in the presence of heparin or high concentrations of inositol 1,4,5-trisphosphate and modulated by compound 48/80, suggesting the involvement of the inositol phospholipid signaling pathway. Latency of appearance and spiking frequency were markedly modulated by varying the intracellular ATP concentration. The differential activation of intracellular calcium signaling and exocytosis by GTP[beta S] confirms the presence of independent signal-transduction pathways for the two cell responses. RP-GTP[beta S] may prove helpful in the biochemical and molecular characterization of Gp, the as-yet-unidentified G protein that couples receptors to intracellular calcium release

Contrast ultrasound for the quantification of deep vein thrombosis in living mice - effects of enoxaparin and P2Y12 receptor inhibition.

BACKGROUND/OBJECTIVES: We examined the applicability of contrast-enhanced ultrasound (CEUS) for imaging of murine deep vein thrombosis (DVT) and measured the effects of enoxaparin, ticagrelor, and P2Y12 receptor deficiency in vivo. METHODS: DVT was induced by exposure to ferric chloride or ligation of the infrarenal vena cava of C57BL/6 mice after pretreatment with enoxaparin, ticagrelor, or vehicle and in P2Y12 -/- mice. Initial thrombus growth was visualized by intravital microscopy. Thrombi were weighed and examined by immunohistochemistry. CEUS was performed with a standard ultrasound system (Vivid 7, GE Healthcare) in the open abdominal cavity after injection of stabilized sulphur hexafluoride microbubbles. RESULTS: Incubation with ferric chloride resulted in non-occluding platelet-containing thrombus growth within 15-25 min. Sham-operated mice, enoxaparin-, and ticagrelor-pretreated wild-type and P2Y12 -/- mice developed only small thrombi. After injection of the contrast agent, growing thrombi delineated clearly as negative contrast in CEUS. Thrombus size in CEUS after 25 minutes was significantly smaller in enoxaparin- (0.3±0.1mm(2) ) and ticagrelor-treated (0.5±0.1mm(2) ) wild-type and in P2Y12 -/- mice (0.4±0.1mm(2) ) as compared to vehicle-treated wild-type mice (2.0±0.3mm(2) ) in the maximal sagittal plane (p < 0.001, n = 5-10). CEUS-derived thrombus size correlated linearly with thrombus weight and also reflected the extent of ligation-induced DVT. CONCLUSIONS: CEUS allowed the real-time quantification of DVT in living mice. Genetic and pharmacologic antithrombotic interventions were well reflected by CEUS and suggested an important role of the platelet P2Y12 receptor in early DVT formation. © 2013 International Society on Thrombosis and Haemostasis.JOURNAL ARTICLEFLWINSCOPUS: ar.jinfo:eu-repo/semantics/publishe

Human thyrotropin receptor gene: expression in thyroid tumors and correlation to markers of thyroid differentiation and dedifferentiation.

Human thyrotropin (TSH) receptor steady-state transcript levels were analyzed by Northern blot analysis in thyroids of patients with thyroid carcinoma, with hyperfunctioning adenoma and in normal controls. In control tissue and benign tumors expression levels of TSH receptor mRNA were high whereas in anaplastic carcinomas no normal TSH receptor mRNA was detected. In papillary and follicular tumors it varied from normal to markedly reduced levels. Thyroid peroxidase (TPO) and thyroglobulin (Tg) mRNA were strongly expressed in normal tissue and in hyperfunctioning adenomas but were completely lost in all anaplastic tumors. In papillary tumors expression of TPO and Tg mRNA varied from normal to a complete loss of expression of either TPO, Tg or both. Tg and TPO steady-state expression did not correlate to TSH receptor transcript levels. C-myc mRNA was highly expressed in anaplastic carcinomas, very variable in normal controls and in differentiated thyroid tumors and low in hyperfunctioning adenomas. In summary, TSH receptor mRNA is persistently expressed in all differentiated thyroid tissues and tumors but lost in undifferentiated carcinomas. Its persistence far along the transformation pathway further supports the concept that this gene which inserts the thyrocytes in the physiological regulatory network is almost constitutively expressed in this cell.Journal ArticleResearch Support, Non-U.S. Gov'tSCOPUS: ar.jinfo:eu-repo/semantics/publishe