RESUMO
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.
Assuntos
Anexina A5/metabolismo , Calcinose/metabolismo , Calgranulina B/metabolismo , Doenças das Artérias Carótidas/metabolismo , Vesículas Citoplasmáticas/metabolismo , Macrófagos/metabolismo , Placa Aterosclerótica/metabolismo , Animais , Apolipoproteínas E/deficiência , Calcinose/patologia , Cálcio/farmacologia , Calgranulina B/genética , Doenças das Artérias Carótidas/patologia , Linhagem Celular , Vesículas Citoplasmáticas/ultraestrutura , Durapatita/metabolismo , Humanos , Macrófagos/ultraestrutura , Macrófagos Peritoneais/fisiologia , Lipídeos de Membrana/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosfatidilserinas/metabolismo , Fósforo/farmacologia , Placa Aterosclerótica/patologia , Interferência de RNA , RNA Interferente Pequeno/farmacologiaRESUMO
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.