Journal: Redox Report : Communications in Free Radical Research

Article Title: Ferroptosis inhibition and mitochondrial rescue: a novel mechanism of emodin in rheumatoid arthritis

doi: 10.1080/13510002.2026.2646383

Figure Lengend Snippet: EMO improves joint histopathological changes and structural damage in CIA mice. (A) EMO's chemical structure. (B) Schematic of the animal experiment. (C) Representative photographs of the hind paws of mice in each group. (D) Representative H&E staining images of ankle joints in each group (200×); red arrow: indicates inflammatory cell infiltration. (E) Representative Safranin O–Fast Green staining images of ankle joints in each group (100×, 200×); blue arrow: indicates cartilage erosion or bone destruction. (F) Representative Micro-CT images of the hind paws of mice in each group; yellow arrows: indicates cartilage erosion or bone destruction. (G–I) Quantitative analyses of Tb.Th, BV/TV and BMD in the hind paw bones of mice (n = 6); data are presented as mean ± standard deviation (SD). ### p < 0.001 vs control group; * p < 0.05, ** p < 0.01, *** p < 0.001 vs the model group. Control group: normal mouse group; Model group: CIA mouse group; EMO-L group: low-dose EMO group, 25 mg/kg; EMO-M group: medium-dose EMO group, 50 mg/kg; EMO-H group: high-dose EMO group, 100 mg/kg; MTX group: methotrexate group, 10 mg/kg; H&E: hematoxylin and eosin staining; TEM: transmission electron microscopy; Tb.Th: trabecular thickness; BV/TV: bone volume/total volume; BMD: bone mineral density.

Article Snippet: Specimens of mouse paws were preserved in 4% paraformaldehyde for a duration of 24 h and subsequently examined with a high-resolution micro-CT system (Skyscan1276, Bruker Corporation, USA).

Techniques: Staining, Micro-CT, Standard Deviation, Control, Transmission Assay, Electron Microscopy

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Comparison of inflammatory-associated miRNA (inflamma-miRNA) profiles in SH- SWE and iNEU- PSEN cells, and in their respective exosomes, relatively to their SH- WT and iNEU- WT control samples. Cells were obtained and differentiated as described in Material and Methods. ( A ) Inflamma-miRNAs expression levels in both cellular AD models reveal that miR-124, miR-125b and miR-21 are increased. ( B ) Representative Transmission Electron Microscopy (TEM) images of exosomes with characteristic sizes after their isolation from cell secretomes by differential ultracentrifugation. ( C ) Representative Western blots for the common exosomal protein markers, Alix, CD63 and Flotilin-1 (FLOT-1). ( D ) Representative histograms of size and concentration of exosomes per sample. ( E ) Profiles of inflamma-miRNA cargo in exosomes from the different mutated cells evidencing increased expression levels of miR-124 and miR-155. Results are mean ± SEM fold change from at least three independent experiments (except NTA single data for exosomes from iNEU cell lines). * p < 0.05 and ** p < 0.01 vs. respective WT levels (dashed lines), two-tailed student’s t test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Comparison, Control, Expressing, Transmission Assay, Electron Microscopy, Isolation, Western Blot, Concentration Assay, Two Tailed Test, Mutagenesis, Derivative Assay, Generated

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Expression levels of miR-124 in SH- WT /SH- SWE and iNEU- WT /iNEU- PSEN cells after transfection with the mock and with the miR-124 inhibitor and mimic. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Schematic representation of the miR-124 modulation with miR-124 inhibitor and/or mimic, as well as with scramble (respective negative controls—NC). ( B ) miR-124 expression levels before and after its modulation in SH- WT /SH- SWE and iNEU- WT /iNEU- PSEN cells. Results are mean ± SEM fold change from at least three independent experiments. ** p < 0.01 vs. respective mock control cells, one-way ANOVA with Bonferroni post-hoc test; # p < 0.05 and ## p < 0.01, between WT and AD mock controls, two-tailed student’s t -test. miR, miRNA; NC, negative control; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Expressing, Transfection, Control, Two Tailed Test, Negative Control, Mutagenesis, Derivative Assay, Generated

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Changes in dendrite outgrowth, mitochondria activation and fusion/fission dynamics by modulation of miR-124 with inhibitor and mimic, relatively to mock, in SH- WT and SH- SWE cells. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Representative fluorescence images of MAP-2 (green) for dendrites, Mitotracker (red) for mitochondria, and MFN-2 (green)/DRP-1 (red) for fusion/fission mitochondria dynamics. Cell nuclei are stained with Hoechst 33,258 dye (blue). Mitochondrial clumps are seen in the neurites (white arrowheads). ( B ) Evaluation of total dendrite length, primary dendrite length and primary dendrite number. ( C ) Quantification of fluorescence intensities for Mitotracker Red, DRP-1 and MFN-2. Results are mean ± SEM fold change, from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. # p < 0.05, between the same treatment in SH- WT and SH- SWE cells, two-tailed student’s t -test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1Δ E9 mutation; MAP-2, microtubule-associated protein 2; DRP1, protein dynamin-related protein 1; MFN-2, mitofusin-2 protein; FI, fluorescence intensity.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Activation Assay, Transfection, Fluorescence, Staining, Two Tailed Test, Expressing, Mutagenesis, Derivative Assay, Generated, Control

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Dendritic spine number decrease in iNEU- WT and iNEU- PSEN cells by modulation with the miR-124 inhibitor, but no changes were noticed for the mimic relatively to the mock transfection. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Representative fluorescence images of MAP-2 (green) staining in axons and dendrites in the different conditions. ( B ) Representative images of high magnification of dendritic segments (from panel A insets) with the miR-124 modulation. ( C ) Quantification of the dendritic spine number in each of the conditions. Results are mean ± SEM from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. miR, miRNA; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1 ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Transfection, Fluorescence, Staining, Derivative Assay, Generated, Control, Mutagenesis

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Effects of miR-124 modulation on APP transcription, Aβ accumulation and Tau phosphorylation in SH- WT /SH- SWE and iNEU- WT /iNEU- PSEN cells. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods. ( A ) Cellular quantification of APP gene expression by RT-qPCR revealing a decrease in the mutated cells with the miR-124 inhibitor. ( B ) Representative Western blots for Aβ species showing different representation and predominance of bands at 120 and 20 kDa. ( C ) Quantification of small and large Aβ oligomers showing that the first are increasingly represented in SH- SWE cells and the later in iNEU- PSEN cells. ( D ) Representative Western blots for Tau phosphorylation status at serine 404. ( E ) Quantification of phospho-Tau/total-Tau ratio revealing a decrease in iNEU-PSEN cells by the miR-124 mimic. Results are mean ± SEM fold change from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. # p < 0.05 and ## p < 0.05, between the same treatment in WT and mutated cells, two-tailed student’s t -test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Phospho-proteomics, Transfection, Gene Expression, Quantitative RT-PCR, Western Blot, Two Tailed Test, Expressing, Mutagenesis, Derivative Assay, Generated, Control

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: miR-124 is transmitted from donor to recipient neurons in a non-cell-contact co-culture system after transfection of miR-124 in SH- WT and SH- SWE donor cells. Cells were obtained, differentiated, and transfected, as detailed in Material and Methods, before being co-cultured with non-transfected matched cells (recipient) for 24 h. ( A ) Schematic representation of SH- WT and SH- SWE donor cells transfected with mock and miR-124 mimic in co-culture with the matched non-transfected recipient cells. ( B ) miR-124 expression levels in the transfected and non-transfected cells after 24 h of co-incubation. Quantification was obtained by RT-qPCR and results are mean ± SEM fold change from at least three independent experiments. ** p < 0.01 vs. mock and # p < 0.05 vs. cells modulated with miR-124 mimic, two-tailed student’s t -test. miR, miRNA; SH-WT, human SH-SY5Y wild-type neurons; SH-SWE, human SH-SY5Y expressing the APP695 Swedish mutant protein.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Co-Culture Assay, Transfection, Cell Culture, Expressing, Incubation, Quantitative RT-PCR, Two Tailed Test, Mutagenesis

Journal: Cells

Article Title: Neuronal Dynamics and miRNA Signaling Differ between SH-SY5Y APPSwe and PSEN1 Mutant iPSC-Derived AD Models upon Modulation with miR-124 Mimic and Inhibitor

doi: 10.3390/cells10092424

Figure Lengend Snippet: Transfection of SH- WT /SH- SWE and of iNEU- WT /iNEU- PSEN cells with miR-124 inhibitor and miR-124 mimic influences miR-124 representation in the vesicle-free secretome (soluble) and packaging into exosomes, while also modifies the representation of other inflamma-miRNAs in the same cells and respective exosomes. ( A ) Evaluation of the influence of the cell treatment with miR-124 inhibitor and miR-124 mimic in the release of miR-124 into the secretome, as a soluble species and as a exosomal cargo. ( B ) Evaluation of cellular inflamma-miRNA expression levels after modulation of miR-124 with its mimic and inhibitor. ( C ) Evaluation of exosomal inflamma-miRNA expression levels after cellular modulation of miR-124 with its mimic and inhibitor. ( D ) Schematic representation of cellular and exosomal inflamma-miRNA distribution in the classical and in the advanced neuronal AD models by miR-124 inhibitor and miR-124 mimic. Results are mean ± SEM fold change from at least three independent experiments. * p < 0.05 and ** p < 0.01 vs. respective mock controls, one-way ANOVA with Bonferroni post-hoc test. miR, miRNA; SH- WT , human SH-SY5Y wild-type neurons; SH- SWE , human SH-SY5Y expressing the APP695 Swedish mutant protein; iNEU- WT , iNeurons derived from induced pluripotent stem cells (iPSCs) generated from a healthy control; iNEU- PSEN , iNeurons from iPSCs generated from a patient carrying the PSEN1ΔE9 mutation.

Article Snippet: Then, miRNA expression was determined by Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR) using the miRCURY LNA TM Universal RT miRNA PCR kit (Qiagen).

Techniques: Transfection, Expressing, Mutagenesis, Derivative Assay, Generated, Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) mRNA and protein expression of S100A9 were measured in human primary macrophages after administration with normal glucose (NG; 5 mmol/L glucose) and high glucose (HG; 25 mmol/L glucose) by real-time quantitative polymerase chain reaction (qPCR) (left, administered for 6 hours, n = 16 PBMC donors) and Western blot (right, administered for 24 hours). These blots from one donor are representative of the 7 PBMC donors. The graph shows the signal intensity of S100A9 / β-actin ratio (n = 7 PBMC donors). (B) S100A9 levels in supernatants of human primary macrophages were measured by ELISA after administration with NG and HG condition for 24 hours (n = 17 PBMC donors). (C) mRNA expression of pro-inflammatory factors, IL-1β, TNF-α and MCP-1, and anti-inflammatory factors, IL-10 and MRC1 was measured in human primary macrophages after stimulation with HG and recombinant human S100A9 (rhS100A9) for 6 hours (n=8 PBMC donors). (D) mRNA expression was measured in human primary macrophages after suppression by S100A9 siRNA for 48 hours and administration with HG for 6 hours (n = 6 PBMC donors). (E) mRNA and protein expression of RAGE were measured in human primary macrophages after stimulation with HG and rhS100A9 by real-time qPCR (left, stimulated for 6 hours, n = 8 PBMC donors) and western blot (right, stimulated for 24 hours). These blots from one donor are representative of the 4 PBMC donors. The graph shows the signal intensity of RAGE / β-actin ratio (n = 4 PBMC donors). (F) mRNA expression of pro-inflammatory factors, IL-1β, TNF-α and MCP-1, and anti-inflammatory factors, IL-10 and MRC1 was measured in human primary macrophages after pretreatment with RAGE-antagonist (FPS-ZM1, 10 μg/mL) for 4 hours, and stimulation with HG and rhS100A9 for 6 hours (n = 6 PBMC donors). P value was calculated by unpaired student’s t-test or one-way ANOVA, based on a comparison with NG, or two-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Enzyme-linked Immunosorbent Assay, Recombinant, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) Size distribution and quantification of EVs derived from human primary macrophages were measured after stimulation with HG and rhS100A9 for 24 hours by nanoparticle tracking analysis (n = 9 PBMC donors). (B) Size distribution and quantification of EVs derived from human primary macrophages were measured after suppression by S100A9 siRNA for 48 hours and administration with NG and HG for 24 hours (n = 7 PBMC donors). (C) Size distribution and quantification of EVs derived from human primary macrophages were measured after pretreatment with RAGE-antagonist (FPS-ZM1, 10 μg/mL) for 4 hours, and stimulation with HG and rhS100A9 for 24 hours (n = 7 PBMC donors). (D) The expression of S100A9 was measured within EVs derived from human primary macrophages by western blotting after stimulation with HG and rhS100A9 for 24 hours. These blots from one donor are representative of the 4 PBMC donors. (E) Calcific potential of EVs derived from human primary macrophages was measured by alkaline phosphatase (ALP) activity after stimulation with HG and rhS100A9 for 24 hours (n = 5 PBMC donors). (F) mRNA expression of the osteogenic factors, BMP2, BMP4, ALP, Runx2, osteopontin, and osteocalcin was measured in human primary macrophages after stimulation with HG and rhS100A9 for 12 hours (n = 8 PBMC donors). (G) mRNA expression of osteogenic factors was measured in human primary macrophages after suppression by S100A9 siRNA for 48 hours and administration with HG for 12 hours (n = 6 PBMC donors). (H) mRNA expression of osteogenic factors was measured in human primary macrophages after pretreatment with RAGE-antagonist (FPS-ZM1, 10 μg/mL) for 4 hours, and stimulation with HG and rhS100A9 for 12 hours (n = 6 PBMC donors). P value was calculated by unpaired student’s t-test or one-way ANOVA, based on a comparison with NG, or two-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Derivative Assay, Expressing, Western Blot, Activity Assay, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) THP-1 cells were cultured in either NG or HG, and then stimulated with rhS100A9 for the indicated times before being analyzed by Western blot with antibodies specific for the indicated targets. Data representative of the 3 different experiments. (B) Nrf2 in a nuclear extract of THP-1 cells (top panel, treated for 24 hours, 4 different experiments) was measured by Western blot and mRNA expression of detoxifying enzymes, hemeoxygenase-1 (HO-1) and NADPH quinone oxidoreductase 1 (Nqo1) (bottom panel, treated for 6 hours) were measured after stimulation with HG and rhS100A9 (n = 4 PBMC donors). (C) The expression of Ser536 phosphorylation of p65 was measured in human primary macrophages by Western blot after pretreatment with VSC2 (10 μM) for 1 hour and stimulation with HG and rhS100A9 for 24 hours. The graph shows the signal intensity of phosphorylated p-65 / p65 ratio (n = 4 PBMC donors). (D) Nrf2 in a nuclear extract of THP-1 cells (top panel, treated for 24 hours, 3 different experiments) was measured by Western blot and mRNA expression of HO-1 and Nqo1 (bottom panel, treated for 6 hours, n = 4 PBMC donors) was measured after pretreatment with VSC2 (10 μM) for 1 hour and stimulation with HG and rhS100A9. (E) mRNA expression of pro-inflammatory factors, IL-1β, TNF-α and MCP-1, and anti-inflammatory factors, IL-10 and MRC1 was measured in human primary macrophages after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 6 hours (n = 4 PBMC donors). (F) The number of EVs derived from human primary macrophages were measured after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 24 hours (n = 4 PBMC donors). (G) mRNA expression of osteogenic factors was measured in human primary macrophages after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 12 hours (n = 4 PBMC donors). (H) Calcific potential of EVs derived from THP-1 cells was measured by ALP activity after pretreatment with VSC2 (10 μM) for 1 hour, and stimulation with HG and rhS100A9 for 24 hours (3 different experiments). P value was calculated by one-way ANOVA, based on a comparison with NG, or two-way ANOVA followed by Bonferroni test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Cell Culture, Western Blot, Expressing, Phospho-proteomics, Derivative Assay, Activity Assay, Comparison

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: Apoe−/− mice fed a HCD for 18 weeks. After 10 weeks, streptozotocin (42 mg/kg/day) was intraperitoneally injected to diabetic group for 5 days, and fed a HCD for 8 weeks. (A) Von Kossa staining on longitudinal sections of the aortic arch; one of 14 animals per group is shown. Scale bars: 200 μm. The graph shows the percentage of positive area in the plaques. (B) Microcalcifications (yellow arrows) within the fibrous cap (white dotted line) were detected by Near-infrared fluorescent (NIRF) calcium tracer (OS680). The graph shows the number of microcalcifications (n=9). Scale bars: 50 μm. (C) Molecular imaging of aortic vascular calcification assessed by OsteoSense750 and vascular inflammation assessed by proteolytic activity, ProSense680. (n=6–10). The graph shows the SUM intensity related to total aortic area for fluorescent reflection imaging (FRI) in Apoe+/+S100a9+/+ (wild type; WT), Apoe−/− S100a9−/− (double knockout; DKO), and Apoe−/− S100a9+/+ (Apoe−/−) mice with diabetes and non-diabetic control. P value was calculated by paired student’s t-test or two-way ANOVA followed by Bonferroni test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Injection, Staining, Imaging, Activity Assay, Double Knockout, Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) Macrophage-targeted LNP containing control siRNA (siControl) or S100a9 siRNA (siS100a9) were injected via tail vein, and F4/80 positive splenic macrophages were isolated. S100A9 mRNA was quantified by real-time PCR. (left panel, n = 3 per each group). Fluorescent in situ hybridization shows less S100A9 mRNA expression localized to macrophage-positive cells in Apoe−/− mice injected with macrophage-targeted LNP containing siS100a9. White arrows show the co-localization of S100A9 mRNA in macrophage-positive cells. One of 4 animals per group is shown (right panel). Scale bars: 20 μm. (B) mRNA expression of S100A9, inflammatory cytokines, and osteogenic factors was measured in splenic macrophages from diabetic and non-diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl (No diabetes and Diabetes) and siS100a9 (Diabetes with siS100a9) (n = 13, No diabetes and Diabetes with siS100a9; n = 16, Diabetes). (C) Immunohistochemical evaluation of ALP activity in aortic plaques from diabetic and non-diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl and siS100a9 (n = 14, No diabetes and Diabetes with siControl; n = 13, No diabetes with siControl; n = 11, Diabetes with siS100a9). Scale bars: 100 μm. (D) In vivo molecular imaging of carotid arteries from diabetic Apoe−/− mice. Vascular calcification assessed by osteogenic activity, OsteoSense680 (red) and vascular inflammation assessed by proteolytic activity, ProSense750 (green). White arrow shows the co-localization of calcification and inflammation, indicating the formation of microcalcification (yellow). The image represents 7–10 mice per group. Scale bar = 200 μm. (E) Molecular imaging of aortic vascular calcification assessed by OsteoSense680 and vascular inflammation assessed by ProSense750. (n= 13–18). The graph shows the SUM intensity related to total aortic area for FRI in diabetic and non-diabetic Apoe−/− mice treated with macrophage-targeted LNP containing control siRNA (siControl) or S100a9 siRNA (siS100a9). (F) 3D micro-CT scanning of aorta from diabetic Apoe−/− mice injected with macrophage-targeted LNP containing siControl and siS100a9 (n = 4, per each group). The graph shows the mean number of microcalcifications assessed by size contribution. P value was calculated by paired student’s t-test or two-way ANOVA followed by Bonferroni test. **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate ± SEM.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Control, Injection, Isolation, Real-time Polymerase Chain Reaction, In Situ Hybridization, Expressing, Immunohistochemical staining, Activity Assay, In Vivo, Imaging, Micro-CT

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: S100A9-RAGE axis accelerates formation of macrophage-mediated extracellular vesicle microcalcification in diabetes

doi: 10.1161/ATVBAHA.118.314087

Figure Lengend Snippet: (A) 25 human atheroma obtained from carotid endarterectomy. Staining for S100A9, RAGE, and ALP in human atheroma (n=25). One of low S100A9 % positive area (left panel) and high S100A9 % positive area (right panel) are shown. Scale bars: 100 μm. Each graph shows the correlation between percentage of S100A9 positive area and ALP- and RAGE-positive area. The average of 5–10 high power fields was used for the analysis. (B) Serial section of human atheroma double-immune stained with anti-S100A9 (red) to anti-RAGE or anti-ALP (green) antibodies. Yellow area in Merge indicate S100A9 positive cells for either RAGE or ALP. Scale bars: 20 μm. The data represent 5 donors that showed similar results. (C) Transmission electron microscopy–based immunogold staining of S100A9 (red arrows) in EVs from human carotid plaques. One of 3 plaques is shown. Scale bar: 300 nm. (D) Carotid plaque specimens were obtained from diabetic (n = 4) and non-diabetic patients (n = 4) undergoing carotid endarterectomy. Volcano plot for the proteomics data. Red and blue markers indicate the significantly enriched (p<0.05) proteins in diabetic patient group and non-diabetic patient group, respectively, with a fold change (FC) cutoff of 2.0. (E) The schematic of the potential mechanism for macrophage-mediated EV microcalcification in diabetes. S100A9-RAGE axis may regulate pro-inflammatory and pro-osteogenic macrophage activation via Nrf2 and NF-κB pathways.

Article Snippet: Confluent macrophages were starved for 24 hours in 0.1% human serum media with 5mM D-glucose (normal glucose condition) or 25mM D-glucose (high glucose condition), and endotoxin-free recombinant human S100A9 (R&D Systems Inc., Minneapolis, MN) was used to stimulate human primary macrophages or the human macrophage-like cell line THP-1 cells.

Techniques: Staining, Transmission Assay, Electron Microscopy, Activation Assay