Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques.

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 areas. We also show that calcification morphology and the plaque's collagen content-two determinants of atherosclerotic plaque stability-are interlinked.

Macrophage-derived matrix vesicles: an alternative novel mechanism for microcalcification in atherosclerotic plaques.

RATIONALE: We previously showed that early calcification of atherosclerotic plaques associates with macrophage accumulation. Chronic renal disease and mineral imbalance accelerate 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. Silencing S100A9 in vitro and genetic deficiency in S100A9-/- mice reduced MV calcification, whereas stimulation with S100A9 increased calcification potential. Externalization of phosphatidylserine after Ca/P stimulation and interaction of S100A9 and annexin V indicated that a phosphatidylserine-annexin V-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 annexin V, which contribute to accelerated microcalcification in chronic renal disease.

Novel endotoxin assay by laser light-scattering particle-counting method.

Exposure of Limulus amoebocyte lysate to endotoxin under stirring produced light-reflective particles that appeared to be coagulin polymers. A laser light-scattering particle counter, the PA-200, detected these particles sensitively. The PA-200 detected endotoxin at a concentration as low as 0.00015 EU/ml in 71 min, whereas the minimum endotoxin concentration measured by a turbidimeter, ET-2000, was 0.0005 EU/ml in 138 min. Moreover, PA-200 was much less affected by the presence of colored substances and refractive materials than was ET-2000. We propose that the high sensitivity, speed, and high interference tolerance of the laser light-scattering particle-counting method make it more useful than the widely used turbidimetric method for quantitative endotoxin assay.

Automated Measurement of Tear Meniscus Height with the Kowa DR-1α Tear Interferometer in Both Healthy Subjects and Dry Eye Patients.

Purpose: To develop and assess a method for quantitation of lower tear meniscus height (TMH) with the Kowa DR-1α 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 ± SD, 50.0 ± 14.0 years) were enrolled. TMH of each subject was measured by two observers both with DR-1α 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-1α was <2.0%. The CV for intraoperator repeatability and interoperator and intersession reproducibility for DR-1α 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 (ß = 0.59, P < 0.001) and ADDE patients (ß = 0.47, P = 0.017). Conclusions: Tear interferometry allows measurement of TMH as reliably as does SS-OCT. DR-1α may inform not only the diagnosis of dry eye disease but also identification of disease subtype.

Quantification of Calcified Particles in Human Valve Tissue Reveals Asymmetry of Calcific Aortic Valve Disease Development.

Recent studies indicated that small calcified particles observable by scanning electron microscopy (SEM) may initiate calcification in cardiovascular tissues. We hypothesized that if the calcified particles precede gross calcification observed in calcific aortic valve disease (CAVD), they would exhibit a regional asymmetric distribution associated with CAVD development, which always initiates at the base of aortic valve leaflets adjacent to the aortic outflow in a region known as the fibrosa. Testing this hypothesis required counting the calcified particles in histological sections of aortic valve leaflets. SEM images, however, do not provide high contrast between components within images, making the identification and quantification of particles buried within tissue extracellular matrix difficult. We designed a new unique pattern-matching based technique to allow for flexibility in recognizing particles by creating a gap zone in the detection criteria that decreased the influence of non-particle image clutter in determining whether a particle was identified. We developed this flexible pattern particle-labeling (FpPL) technique using synthetic test images and human carotid artery tissue sections. A conventional image particle counting method (preinstalled in ImageJ) did not properly recognize small calcified particles located in noisy images that include complex extracellular matrix structures and other commonly used pattern-matching methods failed to detect the wide variation in size, shape, and brightness exhibited by the particles. Comparative experiments with the ImageJ particle counting method demonstrated that our method detected significantly more (p < 2 × 10-7) particles than the conventional method with significantly fewer (p < 0.0003) false positives and false negatives (p < 0.0003). We then applied the FpPL technique to CAVD leaflets and showed a significant increase in detected particles in the fibrosa at the base of the leaflets (p < 0.0001), supporting our hypothesis. The outcomes of this study are twofold: (1) development of a new image analysis technique that can be adapted to a wide range of applications and (2) acquisition of new insight on potential early mediators of calcification in CAVD.

mIMT-visHTS: A novel method for multiplexing isobaric mass tagged datasets with an accompanying visualization high throughput screening tool for protein profiling.

Isobaric mass tagging (IMT) methods enable the analysis of thousands of proteins simultaneously. We used tandem mass tagging reagents (TMT™) to monitor the relative changes in the proteome of the mouse macrophage cell line RAW264.7 at the same six time points after no stimulation (baseline phenotype), stimulation with interferon gamma (pro-inflammatory phenotype) or stimulation with interleukin-4 (anti-inflammatory phenotype). The combined TMT datasets yielded nearly 12,000 protein profiles for comparison. To facilitate this large analysis, we developed a novel method that combines or multiplexes the separate IMT (mIMT) datasets into a single super dataset for subsequent model-based clustering and co-regulation analysis. Specially designed visual High Throughput Screening (visHTS) software screened co-regulated proteins. visHTS generates an interactive and visually intuitive color-coded bullseye plot that enables users to browse the cluster outputs and identify co-regulated proteins.

Angiopoietin Like Protein 2 (ANGPTL2) Promotes Adipose Tissue Macrophage and T lymphocyte Accumulation and Leads to Insulin Resistance.

OBJECTIVES: Angiopoietin-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 FINDINGS: Adenovirus 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. CONCLUSION: ANGPTL2 treatment promotes macrophage accumulation and activation. These results suggest potential mechanisms for insulin resistance.

Uptake of albumin is coupled with stretch-induced hypertrophy of skeletal muscle cells in culture.

Hypertrophy is induced in skeletal muscle when mechanical overload, for example repetitive stretching, is presented. This is a well-known phenomenon and the molecular mechanism involved has been investigated from various aspects. In this study, with a system that enables periodic stretching of cultured skeletal muscle cells, myotubes, along the long cellular axis uni-directionally at a constant frequency, we examined the effects of stretching on skeletal muscle using mouse C2 myotubes in culture as a model. Significant hypertrophy was observed in the myotubes after several days of periodic stretching and this was accompanied by the accumulation of a protein of about 67kDa. This protein was identified with albumin, which was present in the culture medium, based on its antigenicity, size and pI. When bovine serum albumin tagged with biotin was added to the culture medium, it became detectable in the cytoplasm of the stretched myotubes. mRNA encoding albumin was not detectable in the myotubes by northern blotting irrespective of their stretching or non-stretching, indicating that transcription of the albumin gene was not induced in the stretched muscle cells. From these results, we conclude that the accumulation of albumin in stretched myotubes was due to uptake of the protein from the culture medium not to de novo synthesis of the protein in myotubes. We suggest that albumin uptake may be involved in skeletal muscular hypertrophy.

A novel quantitative approach for eliminating sample-to-sample variation using a hue saturation value analysis program.

OBJECTIVES: As computing technology and image analysis techniques have advanced, the practice of histology has grown from a purely qualitative method to one that is highly quantified. Current image analysis software is imprecise and prone to wide variation due to common artifacts and histological limitations. In order to minimize the impact of these artifacts, a more robust method for quantitative image analysis is required. METHODS AND RESULTS: Here we present a novel image analysis software, based on the hue saturation value color space, to be applied to a wide variety of histological stains and tissue types. By using hue, saturation, and value variables instead of the more common red, green, and blue variables, our software offers some distinct advantages over other commercially available programs. We tested the program by analyzing several common histological stains, performed on tissue sections that ranged from 4 µm to 10 µm in thickness, using both a red green blue color space and a hue saturation value color space. CONCLUSION: We demonstrated that our new software is a simple method for quantitative analysis of histological sections, which is highly robust to variations in section thickness, sectioning artifacts, and stain quality, eliminating sample-to-sample variation.

Simplified preparation of crude and functional coagulogen by thermal inactivation of serine proteases in Limulus amebocyte lysate and its application for rapid endotoxin determination.

The effects of thermal treatment on Limulus amebocyte lysate (LAL) reagent were studied. Thermal resistances of enzymes and coagulogen in LAL reagent were evaluated by aggregometry and SDS-PAGE. Although enzyme activities of LAL reagent were completely lost after heating at temperatures above 60 °C for 10 min, gelating activities of coagulogen were retained even over 80 °C. Phenylmethanesulfonyl fluoride (PMSF; 1 mmol/mL), a strong non-specific serine-protease inhibitor, did not completely inactivate serine-protease activities of LAL. As a result, complete hydrolysis of coagulogen to coagulin was unexpectedly obtained. Solvent treatment of LAL was similar in effect to thermal treatment of LAL, but there were 2 problems: complete removal of solvent from samples and increased solution turbidity during preparation. To study the application of thermal-treated LAL, we conjugated it with titania particles. LAL-conjugated titania particles were obtained as small aggregates between titania nanoparticles and thermal-treated LAL (LAL-conjugated microbeads; LCM). When the mixture of LCMs and fresh LAL reagent was reacted with endotoxin an acute aggregation of LCMs was induced prior to the aggregate formation of LAL as monitored by stirring turbidimetry. This method, endotoxin microbeads aggregometry (EMA) may provide a rapid and sensitive method for endotoxin determination.

Turbidimetric method for evaluation of photocatalytic activities of suspended fine particles.

A spectrophotometer with special cuvette was developed for evaluating the photocatalytic activities of suspended fine particles. The spectrophotometer can continuously irradiate UV light using LED to the sample solution, and changes in the absorbance at 664 nm during photocatalytic degradation of methylene blue (MB) were monitored continuously. From the onset of MB degradation, the absorbance decreased and reached a steady value at the end of the reaction. This process was expressed by first order kinetics and the photocatalytic activities of various fine particles could be evaluated quantitatively based on the reaction rate constant (k). The effect of photocatalysis using various TiO2 fine particles on the physiological activities of Euglena gracilis was related with k value.

Determination of lead(II) in fly ash leachate using a newly developed simple spectrophotometric method.

A new simple method for the spectrophotometric determination of Pb(II) in fly ash leachates was developed. These leachates tend to contain a large amount of Ca(II) and Zn(II); this interferes with spectrophotometric determination of Pb(II) when conventional colorimetric agents are used. A copolymer consisting of protoporphyrin IX disodium salt and acrylamide was synthesized as a colorimetric agent. A measuring reagent containing ethylenediamine-N,N'-dipropionic acid (EDDP) as a masking agent for Zn(II) and an appropriate amount of Ca(II) together with the copolymer was applied to determine Pb(II). The temporal change in the absorption spectrum of the measuring reagent was acquired with a newly developed portable spectrophotometer for this method. The composition of EDDP and Ca(II) in the measuring reagent was optimized to measure leachates contaminated with Ca(II) and Zn(II). The detection limit and relative standard deviation of Pb(II) measured using the optimized method were 0.05 mg L(-1) and 2.3%, respectively. The tolerance limits for Ca(II) and Zn(II) contaminants, where errors of less than 10% were allowed at a concentration of 0.5 mg L(-1) Pb(II), were 4000 and 4 mg L(-1), respectively. The determination of Pb(II) in various samples of actual leachates from incinerator fly ash was examined with this method. The obtained values correlated well with those obtained by flame atomic absorption spectroscopy.

Classification of pollen species using autofluorescence image analysis.

A new method to classify pollen species was developed by monitoring autofluorescence images of pollen grains. The pollens of nine species were selected, and their autofluorescence images were captured by a microscope equipped with a digital camera. The pollen size and the ratio of the blue to red pollen autofluorescence spectra (the B/R ratio) were calculated by image processing. The B/R ratios and pollen size varied among the species. Furthermore, the scatter-plot of pollen size versus the B/R ratio showed that pollen could be classified to the species level using both parameters. The pollen size and B/R ratio were confirmed by means of particle flow image analysis and the fluorescence spectra, respectively. These results suggest that a flow system capable of measuring both scattered light and the autofluorescence of particles could classify and count pollen grains in real time.