The Scavenger Receptor CD68 Regulates Platelet Mediated Oxidized Low-Density Lipoprotein (oxLDL) Deposition in Atherosclerotic Vessels at an Early Stage of Atherosclerosis in LDLR-/-/ApoBec-/- Mice.

BACKGROUND/AIMS: Oxidative modifications of low-density lipoprotein (ox-LDL) play a key role in initial steps of atheroprogression possibly via specific scavenger receptors on inflammatory and endothelial cells. Amongst others, CD68 might play a crucial role in this leading to fatty streak formation. METHODS: Different CD68-Fc fusion proteins were cloned, expressed and tested in vitro for their oxLDL binding properties as a decoy for endogenous oxLDL. Physiological functions were tested in foam cell assays with human monocytes in culture and by binding oxLDL from human blood. The best suited candidate FcIgG2-FL-CD68 was injected twice weekly in LDL receptor and ApoBec deficient mice (LDLR-/-/Apobec-/-), and the oxLDL content was measured in peripheral blood, in different cell types of the spleen and aortic wall by specific oxLDL antibodies using flow cytometry. RESULTS: Different variants of the CD68-Fc bound to copper-oxided LDL (oxLDL), LDL and to a lesser extent HDL with different efficacy in an ELISA based binding assay in vitro. Native oxLDL content in human blood derived from patients with extended atherosclerosis was reduced after passage through a specific protein G column conjugated with the different CD68-Fc fusion proteins. Foam cell formation from human peripheral blood monocyte-platelet co-culture was reduced by the most effective CD68-Fc fusion proteins. oxLDL was not increased in the blood but markedly increased in the vessel wall from LDLR-/-/Apobec-/- mice at an early stage of atherosclerosis. Platelet-like cells in the vessel well contributed most to the increase in tissue oxLDL. FcIgG2-FL-CD68, reduced oxLDL content of aortic vessel wall cells from LDLR-/-/Apobec-/- mice. However a tissue specific reduction on the oxLDL content in peripheral blood, the spleen or cells from the aortic vessel by FcIgG2-FL-CD68 could not be shown. CONCLUSION: Platelets contribute to increased tissue oxLDL in the aortic wall but not in peripheral blood. CD68 seems to play a role in the oxLDL metabolism in the vessel wall at early stages of atherosclerosis. FcIgG2-FL-CD68 could serve as a novel therapeutic option to modify the oxLDL content in the vessel wall.

Mapping Thyroid Changes in Size and Position During Enlargement in Adult Mice With Hyperthyroidism.

The thyroid in Graves' disease undergoes a considerable divergence in size and position from the normal anatomy. However, knowledge of the pathological anatomy related to the change, which is required before planned surgical or local intervention, or diagnosis, is neglected. To investigate Graves' disease, we established a model of mice that successfully mimicked all the signs presented in the clinic. Under a long-term immunization (35 weeks), the animals displayed large heterogeneity in thyroid size, such as the cases of natural occurrence. These thyroids in the model were sized into various phases and registered. A blend of the registered thyroids and the thyroid and tracheal cartilage landmarks led to the production of site-dependent incidence graphs of thyroid in the front view and on the section for each phase. The merger of the incidence graphs of all the phases resulted in thyroid phase-dependent topography. The depicted graphs illustrate the fine localization of the thyroid in various sizes and their dynamic changes during enlargement, which may facilitate currently used fine-needle aspiration biopsy and ultrasonography-guided biopsy techniques. Familiarity with this knowledge might avoid misclassifying an abnormality as normal, or vice versa, and be helpful for imaging diagnosis and local surgery therapy in Graves' disease.

Flow chamber staining modality for real-time inspection of dynamic phenotypes in multiple histological stains.

Traditional histological stains, such as hematoxylin-eosin (HE), special stains, and immunofluorescence (IF), have defined myriads of cellular phenotypes and tissue structures in a separate stained section. However, the precise connection of information conveyed by the various stains in the same section, which may be important for diagnosis, is absent. Here, we present a new staining modality-Flow chamber stain, which complies with the current staining workflow but possesses newly additional features non-seen in conventional stains, allowing for (1) quickly switching staining modes between destain and restain for multiplex staining in one single section from routinely histological preparation, (2) real-time inspecting and digitally capturing each specific stained phenotype, and (3) efficiently synthesizing graphs containing the tissue multiple-stained components at site-specific regions. Comparisons of its stains with those by the conventional staining fashions using the microscopic images of mouse tissues (lung, heart, liver, kidney, esophagus, and brain), involving stains of HE, Periodic acid-Schiff, Sirius red, and IF for Human IgG, and mouse CD45, hemoglobin, and CD31, showed no major discordance. Repetitive experiments testing on targeted areas of stained sections confirmed the method is reliable with accuracy and high reproducibility. Using the technique, the targets of IF were easily localized and seen structurally in HE- or special-stained sections, and the unknown or suspected components or structures in HE-stained sections were further determined in histological special stains or IF. By the technique, staining processing was videoed and made a backup for off-site pathologists, which facilitates tele-consultation or -education in current digital pathology. Mistakes, which might occur during the staining process, can be immediately found and amended accordingly. With the technique, a single section can provide much more information than the traditional stained counterpart. The staining mode bears great potential to become a common supplementary tool for traditional histopathology.

Minimal Collagen-Binding Epitope of Glycoprotein VI in Human and Mouse Platelets.

Glycoprotein VI (GPVI) is a platelet-specific receptor for collagen and fibrin, regulating important platelet functions such as platelet adhesion and thrombus growth. Although the blockade of GPVI function is widely recognized as a potent anti-thrombotic approach, there are limited studies focused on site-specific targeting of GPVI. Using computational modeling and bioinformatics, we analyzed collagen- and CRP-binding surfaces of GPVI monomers and dimers, and compared the interacting surfaces with other mammalian GPVI isoforms. We could predict a minimal collagen-binding epitope of GPVI dimer and designed an EA-20 antibody that recognizes a linear epitope of this surface. Using platelets and whole blood samples donated from wild-type and humanized GPVI transgenic mice and also humans, our experimental results show that the EA-20 antibody inhibits platelet adhesion and aggregation in response to collagen and CRP, but not to fibrin. The EA-20 antibody also prevents thrombus formation in whole blood, on the collagen-coated surface, in arterial flow conditions. We also show that EA-20 does not influence GPVI clustering or receptor shedding. Therefore, we propose that blockade of this minimal collagen-binding epitope of GPVI with the EA-20 antibody could represent a new anti-thrombotic approach by inhibiting specific interactions between GPVI and the collagen matrix.

Definition of a sectioning plane and place for a section containing hoped-for regions using a spare counterpart specimen.

Histological examination of targets in regions of interest in histological sections is one of the most frequently used tools in biomedical research. However, it is a technical challenge to secure a multitarget section for inspection of the structure's mutual relationship of targets or a longitudinally filamentous- or tubular-formed tissue section for visitation of the overall morphological features. We present a method with a specified cutting plane and place, allowing researchers to cut directly at the multitarget centers accurately and quickly. The method is proven to be reliable with high accuracy and reproducibility and a low coefficient of variation, testing on repeat experiments of three target's position-known models. With this method, we successfully yielded single sections containing whole intraorbital optical nerves, three aortic valves, or whole thoracic tracheas in their central positions. The adjoined custom-made tools used in the study, such as various tissue-specific formulated calibrated trimming and embedding guides, an organ-shaped cavity plaster mold, and a two-time embedding technique for optimal and identical trimming or embedding, also bear great potential to become a common supplemental tool for traditional histology and may contribute to the reduction of the labor, and the number of animals needed.

Comprehensive Characterization of Platelet-Enriched MicroRNAs as Biomarkers of Platelet Activation.

Dysregulation of platelet function is causally connected to thrombus formation and cardiovascular diseases. Therefore, assessing platelet reactivity is crucial. However, current platelet function tests come with pitfalls, limiting clinical use. Plasma miRNA signatures have been suggested as novel biomarkers for predicting/diagnosing cardiovascular diseases and monitoring antiplatelet therapy. Here, we provide results from a comprehensive study on the feasibility of using circulatory platelet miRNAs as surrogate markers of platelet activation. We performed small RNA-Seq on different blood cell types to confirm known and identify novel platelet-enriched miRNAs and validated a panel of 16 miRNAs using RT-qPCR. To identify the main carrier of these blood-based platelet miRNAs, we enriched and analyzed distinct microvesicle populations. Platelets were stimulated with GPVI and P2Y12 agonists in vitro to monitor the release of the selected miRNAs following activation. Finally, the miRNA panel was also measured in plasma from mice undergoing the Folts intervention (recurrent thrombus formation in the carotid artery). Applying an unbiased bioinformatics-supported workflow to our NGS data, we were able to confirm a panel of previously established miRNA biomarker candidates and identify three new candidates (i.e., miR-199a-3p, miR-151a-5p, and miR-148b-3p). Basal levels of platelet-derived miRNAs in plasma were mainly complexed with proteins, not extracellular vesicles. We show that changes in miRNA levels due to platelet activation are detectable using RT-qPCR. In addition, we highlight limitations of studying the in vitro release of miRNAs from platelets. In vivo thrombosis resulted in significant elevations of platelet-derived miRNA levels in mice. In conclusion, we provide in-depth evidence that activated platelets release miRNAs, resulting in measurable changes in circulatory miRNA levels, rendering them promising biomarker candidates.

Histo-ELISA technique for quantification and localization of tissue components.

A novel Histo-ELISA technique is intended to facilitate quantification of target tissue proteins in a tissue section and involves the selection of target regions in the tissue section, application of streptavidin-conjugated HRP (horseradish peroxidase), coupled with peroxidase substrate-TMB (3,3',5,5'-tetramethylbenzidine), and staining dye evaluation with ELISA reader. The target protein content (weight per volume unit) was translated from optical densities by a reference standard curve, obtained via parallel staining of the targeted protein-coated slides. To validate the technique, we carried out quantifications of IgG extravasation in ischemic and nonischemic brain sections in a mouse stroke model. With those obtained data and the reference of immunohistochemistry scores assessed on the adjacent sections, accuracy, sensitivity, and precision for the technique were evaluated. For all evaluated parameters, Histo-ELISA performance was either comparable to or better than the standard immunohistochemistry. A comparison with the data from the repeated measurements yielded a rather low coefficient of variation. The results confirmed that the technique is a fairly reliable quantitative test with rather high sensitivity, accuracy, precision, and reproducibility for detecting target protein content in tissue sections and that its tissue distribution and related subsequent morphological changes can be observed at the same time.

Combined administration of the GPVI-Fc fusion protein Revacept with low-dose thrombolysis in the treatment of stroke.

BACKGROUND: Thrombolytic therapy with recombinant tissue plasminogen activator (rtPA) remains the only approved medication for acute ischemic stroke, but incurs significant bleeding risks. Therefore, approaches to combine lower doses of thrombolytic therapy with other effective drugs aim at improving efficacy and reducing bleeding rates. We examined the safety and therapeutic effects of various dosings of rtPA, either alone or combined with glycoprotein VI-Fc fusion protein (GPVI-Fc, Revacept) on experimental stroke in mice. METHODS AND RESULTS: The effect of filament-induced intracerebral thrombus formation and embolization was investigated after a one-hour occlusion of the middle cerebral artery. In accordance with previous studies, treatment with 10 mg/kg rtPA significantly improved functional outcome, cerebral infarct size and edema, but also resulted in markedly increased intracranial bleeding volumes. In contrast, low doses of rtPA (0.1 or 0.35 mg/kg body weight) did not change outcome parameters. However, addition of 1 mg/kg Revacept to 0.35 mg/kg rtPA led to improved reperfusion compared to rtPA alone. Moreover, these combined treatments resulted in improved grip strength, compared to the respective dose of rtPA alone. Infarct-surrounding edema improved after combined treatments, but not after respective single rtPA dosings. Intracranial bleeding volumes were below controls after all low-dose rtPA therapies, given either alone or combined with Revacept. CONCLUSIONS: In contrast to using the equally effective full dose of rtPA, intracranial bleeding was not increased by low-dose rtPA combined with Revacept. Therefore, addition of Revacept to low-dose rtPA does not incur safety risks, but improves efficacy of treatment.

The GPVI-Fc fusion protein Revacept improves cerebral infarct volume and functional outcome in stroke.

OBJECTIVES: We examined the effect of Revacept, an Fc fusion protein which is specifically linked to the extracellular domain of glycoprotein VI (GPVI), on thrombus formation after vessel wall injury and on experimental stroke in mice. BACKGROUND: Several antiplatelet drugs for the treatment of myocardial infarction or ischemic stroke with potent anti-ischemic effects have been developed, but all incur a significant risk of bleeding. METHODS: Platelet adhesion and thrombus formation after endothelial injury was monitored in the carotid artery by intra-vital fluorescence microscopy. The morphological and clinical consequences of stroke were investigated in a mouse model with a one hour-occlusion of the middle cerebral artery. RESULTS: Thrombus formation was significantly decreased after endothelial injury by 1 mg/kg Revacept i.v., compared to Fc only. 1 mg/kg Revacept i.v. applied in mice with ischemic stroke immediately before reperfusion significantly improved functional outcome, cerebral infarct size and edema compared to Fc only. Also treatment with 10 mg/kg rtPA was effective, and functional outcome was similar in both treatment groups. The combination of Revacept with rtPA leads to increased reperfusion compared to treatment with either agent alone. In contrast to rtPA, however, there were no signs of increased intracranial bleeding with Revacept. Both rtPA and Revacept improved survival after stroke compared to placebo treatment. Revacept and vWF bind to collagen and Revacept competitively prevented the binding of vWF to collagen. CONCLUSIONS: Revacept reduces arterial thrombus formation, reduces cerebral infarct size and edema after ischemic stroke, improves functional and prognostic outcome without intracranial bleeding. Revacept not only prevents GPVI-mediated, but probably also vWF-mediated platelet adhesion and aggregate formation. Therefore Revacept might be a potent and safe tool to treat ischemic complications of stroke.

The GPVI-Fc fusion protein Revacept reduces thrombus formation and improves vascular dysfunction in atherosclerosis without any impact on bleeding times.

AIMS: Glycoprotein VI (GPVI) is a key platelet receptor which mediates plaque-induced platelet activation and consecutive atherothrombosis, but GPVI is also involved in platelet-mediated atheroprogression. Therefore, interference in GPVI-mediated platelet activation has the potential to combine short-term and long-term beneficial effects, specificity and safety especially regarding bleeding complications. METHODS AND RESULTS: We investigated the effects of the soluble dimeric GPVI receptor fusion protein, Revacept, an antagonist of collagen-mediated platelet activation, in an animal model of atherosclerosis: twenty week old rabbits, which had been fed on a cholesterol-rich diet for 8 weeks, received Revacept (8 mg/kg) or control twice weekly for 4 weeks. Pharmacokinetics indicated a slight accumulation of the drug in the serum after repeated dosing of Revacept for 3 weeks. A significant improvement of endothelial dysfunction after 0.06 and 0.6 µg/min acetylcholine and a significant decrease of vessel wall thickening were found after Revacept treatment. Accordingly, aortic vessel weight was reduced, and plaque sizes, macrophage and T-cell invasion tended to be reduced in histological evaluations. Bleeding time was determined after tail clipping in mice. Revacept alone or in combination with widely used anti-platelet drugs revealed a high safety margin with no prolongation of bleeding times. CONCLUSION: Repeated doses of Revacept led to a significant improvement of endothelial dysfunction and vascular morphology in atherosclerotic rabbits. Furthermore, no influence of Revacept on bleeding time alone or in combinations with various anti-platelet drugs was found in mice. Thus, the inhibition of collagen-mediated platelet interaction with the atherosclerotic endothelium by Revacept exerts beneficial effects on morphology and vascular function in vivo and seems to have a wide therapeutic window without influencing the bleeding time.