Clinical evidence links arterial calcification and cardiovascular risk. Finite-element modelling of the stress distribution within atherosclerotic plaques has suggested that subcellular microcalcifications in the fibrous cap may promote material failure of the plaque, but that large calcifications can stabilize it. Yet the physicochemical mechanisms underlying such mineral formation and growth in atheromata remain unknown. Here, by using three-dimensional collagen hydrogels that mimic structural features of the atherosclerotic fibrous cap, and high-resolution microscopic and spectroscopic analyses of both the hydrogels and of calcified human plaques, we demonstrate that calcific mineral formation and maturation results from a series of events involving the aggregation of calcifying extracellular vesicles, and the formation of microcalcifications and ultimately large calcification zones. We also show that calcification morphology and the plaque’s collagen content – two determinants of atherosclerotic plaque stability - are interlinked.

Rationale We previously showed that early calcification of atherosclerotic plaques associates with macrophage accumulation. Chronic renal disease (CRD) and mineral imbalance accelerates calcification and the subsequent release of matrix vesicles (MVs) — precursors of microcalcification. Objective We tested the hypothesis that macrophage-derived MVs contribute directly to microcalcification. Methods and Results Macrophages associated with regions of calcified vesicular structures in human carotid plaques (n=136 patients). In vitro, macrophages released MVs with high calcification and aggregation potential. MVs expressed exosomal markers (CD9 and TSG101), and contained S100A9 and annexin V (Anx5). Silencing S100A9 in vitro and genetic deficiency in S100A9−/− mice reduced MV calcification, while stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine (PS) after Ca/P stimulation and interaction of S100A9 and Anx5, indicated that a PS-Anx5-S100A9 membrane complex facilitates hydroxyapatite nucleation within the macrophage-derived MV membrane. Conclusions Our results support the novel concept that macrophages release calcifying MVs enriched in S100A9 and Anx5, which contribute to accelerated microcalcification in CRD.

Recombinant thrombopoietin has been reported to stimulate megakaryocytopoiesis and thrombopoiesis and it may be quite useful to treat patients with low platelet counts after chemotherapy. As little is known regarding the possible activation of platelets by thrombopoietin, we examined the effects of thrombopoietin on platelet aggregation induced by shear stress and various agonists in native plasma. Using hirudin as an anticoagulant, thrombopoietin (1 to 100 ng/mL) enhanced platelet aggregation induced by 2 micromol/L adenosine- diphosphate (ADP) in a dose dependent fashion. The enhancement was not affected by treatment of platelets with 1 mmol/L aspirin plus SQ-29548 (a thromboxane antagonist, 1 micromol/L) but was inhibited by a soluble form of the thrombopoietin receptor, suggesting that the enhancement was mediated by the specific receptors and does not require thromboxane production. Epinephrine (1 micromol/L), which does not induce platelet aggregation in hirudin platelet rich plasma (PRP), did so in the presence of thrombopoietin (10 ng/mL). Thrombopoietin (10 ng/mL) also enhanced or primed platelet aggregation induced by collagen (0.5 micron.mL),. thrombin, serotonin, and vasopressin. Thrombopoietin does not induce any rise in cytosolic ionized calcium concentration nor activation of protein kinase C, as estimated by phosphorylation of preckstrin, indicating that the priming effects of thrombopoietin does not require those processes. The ADP- or thrombin-induced rise in cytosolic ionized calcium concentration was not enhanced by thrombopoietin (100 ng/mL). Further, shear (ca. 90 dyn/cm2)-induced platelet aggregation was also potentiated by thrombopoietin. The priming effect on epinephrine-induced platelet aggregation in hirudin PRP was unique to thrombopoietin, with no effects seen using interleukin-6 (IL-6), IL-11, IL-3, erythropoietin, granulocyte-colony stimulating factor, granulocyte macrophage-colony stimulating factor, or c-kit ligand. These data indicate that monitoring of platelet functions may be necessary in the clinical trials of thrombopoietin.

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ObjectivesAngiopoietin-like protein 2 (ANGPTL2), a recently identified pro-inflammatory cytokine, is mainly secreted from the adipose tissue. This study aimed to explore the role of ANGPTL2 in adipose tissue inflammation and macrophage activation in a mouse model of diabetes.Methodology/Principal FindingsAdenovirus mediated lacZ (Ad-LacZ) or human ANGPTL2 (Ad-ANGPTL2) was delivered via tail vein in diabetic db/db mice. Ad-ANGPTL2 treatment for 2 weeks impaired both glucose tolerance and insulin sensitivity as compared to Ad-LacZ treatment. Ad-ANGPTL2 treatment significantly induced pro-inflammatory gene expression in white adipose tissue. We also isolated stromal vascular fraction from epididymal fat pad and analyzed adipose tissue macrophage and T lymphocyte populations by flow cytometry. Ad-ANGPTL2 treated mice had more adipose tissue macrophages (F4/80+CD11b+) and a larger M1 macrophage subpopulation (F4/80+CD11b+CD11c+). Moreover, Ad-ANGPTL2 treatment increased a CD8-positive T cell population in adipose tissue, which preceded increased macrophage accumulation. Consistent with our in vivo results, recombinant human ANGPTL2 protein treatment increased mRNA levels of pro-inflammatory gene products and production of TNF-α protein in the human macrophage-like cell line THP-1. Furthermore, Ad-ANGPTL2 treatment induced lipid accumulation and increased fatty acid synthesis, lipid metabolism related gene expression in mouse liver.ConclusionANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance.

We developed a novel flow particle analyzer that automatically classifies airborne pollen grains. The design of the particle counter (model KP-1000) is based on that of a flow cytometer, applied to the measurement of airborne particles. The counter classifies pollen species by simultaneously detecting both scattered light and the characteristic fluorescence excited by ultraviolet light in the flow cell. We observed airborne pollen using KP-1000 pollen counters and Durham samplers to compare their performance at three study sites in Japan during the spring pollen season. The pollen counter followed the variation in pollen concentrations, and its daily pollen counts were significantly correlated with the results of the Durham sampling method at all study sites. Although the counter over-or under-counted 2 target pollen species (Cryptomeria japonica and Chamaecyparis obtusa) when they coexisted, a data correction based on the Durham sampling results improved the accuracy of pollen classification of the counter. Our results indicate that the new pollen counter has a strong potential for counting and identifying airborne pollen grains in real time, and it requires further improvement, field trials, and tests with other common airborne pollen grains.

Mechanical stress causes various responses in cells both in vivo and in vitro. Realignment of cells and stress fibers is one of the remarkable phenomena that are induced by the stress. However, the mechanism by which their realignment is controlled is largely unknown. In this study, effects of mechanical stretch on the morphology of cultured cells were examined using a cyclic and reciprocal cell stretching apparatus. A10 cells, a cell line derived from rat aortic smooth muscle, were used as a model, since they are spindle-shaped and have remarkable stress fibers aligned along the longitudinal cell axis. Therefore, the orientation of the cell and stress fibers could be easily identified. When the cells were cultured on elastic silicone membranes and subjected to cyclic and reciprocal stretch with an amplitude of 20% at a frequency of 60 cycles per minute, actin stress fibers were aligned obliquely to the direction of stretching with angles of 50 to 70 degrees within about 15 min after the onset of stretching. Then, after 1-3 hr of cyclic stretching, the long axes of a majority of the cells were also reoriented to similar directions to the stress fibers. The stretchinduced cell reorientation was blocked by 1 µM cytochalasin B, but not by colcemid. These results indicate that the orientation of cells and actin filaments are closely related and actin filaments play a critical role in the early step of the cell reorientation.

PURPOSE. To develop and assess a method for quantitation of lower tear meniscus height (TMH) with the Kowa DR-1a tear interferometer. METHODS. Sixty-nine eyes of 49 men and 20 women (36 healthy volunteers, 33 patients with aqueous-deficient dry eye [ADDE]; mean age 6 SD, 50.0 6 14.0 years) were enrolled. TMH of each subject was measured by two observers both with DR-1a and newly developed software and with anterior-segment swept-source optical coherence tomography (SS-OCT). Intraoperator repeatability and interoperator and intersession reproducibility of measurements were assessed based on the within-subject SD (Sw), coefficient of variation (CV), and intraclass correlation coefficient (ICC). Agreement between the two devices was assessed by regression and Bland-Altman analysis. RESULTS. The CV for system repeatability of DR-1a was <2.0%. The CV for intraoperator repeatability and interoperator and intersession reproducibility for DR-1a measurements was 9.6%, 4.5%, and 4.4% in healthy subjects, respectively, and 16.8%, 9.8%, and 10.3% in ADDE patients. All corresponding ICC values were ‡0.87 in healthy subjects and ‡0.48 in ADDE patients. Bland-Altman plots indicated a high level of agreement between the two devices. Schirmer test value was significantly correlated with interferometric TMH in both healthy subjects (b ¼ 0.59, P < 0.001) and ADDE patients (b ¼ 0.47, P ¼ 0.017). CONCLUSIONS. Tear interferometry allows measurement of TMH as reliably as does SS-OCT. DR-1a may inform not only the diagnosis of dry eye disease but also identification of disease subtype.