Journal: Scientific Reports

Article Title: Endothelial Cell Phenotypes are Maintained During Angiogenesis in Cultured Microvascular Networks

doi: 10.1038/s41598-018-24081-z

Figure Lengend Snippet: Illustration of CD36 labeling in ex vivo cultured microvascular networks and stimulated in vivo microvasculature. Comparison between Day 3 ( Ex Vivo ) ( A–C ) and Day 3 ( In Vivo ) ( D–F ) microvascular networks revealed diminishing CD36 labeling along the length of the capillary sprout becoming absent at the tip. Arrows identify examples of CD36-negative capillary sprouts. Scale bars = 200 µm. ( G , H ) Capillary sprouts exhibited a substantial decrease in CD36 labeling compared to larger network vessels ( > 10 µm) in Day 3 ( Ex Vivo ) and Day 3 ( In Vivo ) tissues. White and black bars represent “Sprouts” and “Networks” respectively for Day 3 ( Ex Vivo ) and Day 3 ( In Vivo ) groups. The *** indicates a significant difference of p < 0.001 by Mann-Whitney U test.

Article Snippet: The following primary antibodies were used: UNC5b (1:100; Abcam; Cambridge, MA), VEGFR-2 (1:50; Santa Cruz Biotechnology; Dallas, TX), Alexa-568 Phalloidin (1:50; Invitrogen; Carlsbad, CA), CD36 (1:100; Novus Biologicals; Littleton, CO), and PECAM (1:200; BD Pharmingen; San Jose, CA).

Techniques: Labeling, Ex Vivo, Cell Culture, In Vivo, Comparison, MANN-WHITNEY

Journal: Cell Death & Disease

Article Title: Microparticles from apoptotic platelets promote resident macrophage differentiation

doi: 10.1038/cddis.2011.94

Figure Lengend Snippet: Differentiation of monocytes into resident phagocytic cells induced by PM ap . ( a ) Surface spreading and actin cytoskeleton rearrangements of THP-1 cells, cultured for 7 days with vehicle buffer or PM ap . Bright-field images indicate cell spreading and filopod formation (arrows); images of FITC-phalloidin fluorescence show strands of actin filaments with PM ap (scale bars: 100 μ m, representative for three experiments). ( b ) Proliferation of THP-1 cells after 7 days of incubation with vehicle or PM ap . Data are expressed as absolute cell numbers. ( c ) Relative amount (%) of necrotic cells after incubation with PMA (10 ng/ml), PM ap , or resting platelets. ( d ) Uptake of oxLDL (labeled with DiI) by THP-1 cells, incubated for 7 days with PM ap or resting platelets (plts). ( e and f ,) Expression of phagocytic cell markers on THP-1 cells ( e ) and primary monocytes ( f ). Cells were treated for 2 or 7 days with vehicle or PM ap . Surface expression of CD36 was determined by flow cytometry, as was the intracellular expression of CD68 in permeabilized cells. Incubation conditions were as described for . Mean±S.E.M. ( n =4–5). * P <0.05 versus control

Article Snippet: THP-1 cells or monocytes in suspension were stained (30 min, 4 °C) with mouse anti-human antibodies against CD11b, CD31 (Sigma, St. Louis, MO, USA); CD14, CD16, CCR2, CCR5, CXCR4 (BD Biosciences); or CD36 (ImmunoTools, Friesoythe, Germany) and prepared as described before.

Techniques: Cell Culture, Fluorescence, Incubation, Labeling, Expressing, Flow Cytometry

Journal: Hepatology Communications

Article Title: Hyperoxidized Albumin Modulates Platelets and Promotes Inflammation Through CD36 Receptor in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1440

Figure Lengend Snippet: HNA2 induces platelet aggregation and activation. (A) Platelets were incubated with ex vivo prepared HMA (1 mg/mL), HNA1 (1 mg/mL), and HNA2 (1 mg/mL), and aggregation was induced with collagen (0.2 μg/mL). Representative reading and bar graph indicate increased aggregation in HNA2‐treated platelets as compared with HNA1 and HMA. Data are expressed as percent of normalized platelet aggregation (n = 3). (B) CD40L and ROS production in healthy platelets was significantly increased when exposed to HNA2 (1 mg/mL). Pretreatment with anti‐CD36 blocking antibody (1 μg/mL) significantly reverses the effect, particularly in HNA2‐exposed platelets. (C) EGTA decreases HNA2‐induced platelet expression of CD40L and ROS production in a dose‐dependent manner. Platelets were pre‐incubated with EGTA, and activation was induced with HNA2 (1 mg/mL). (D) Histogram represents the percent expression of platelets exposed to HNA2 (1 mg/mL) showing increased expression of P‐selectin, PAC‐1, and intracellular calcium as compared with HNA1 (n = 5). (E) Expression of SNARE complex proteins (Syntaxin‐11, SNAP‐23, and VAMP‐8) increases in healthy platelets exposed to HNA2 (1 mg/mL) as compared with HNA1 and HMA. Interestingly, anti‐CD36 blocking antibody (1 μg/mL) reduced the effect of HNA2 over others. Results expressed as mean ± SEM (* P < 0.05, ** P < 0.01, *** P < 0.001).

Article Snippet: Platelets were pretreated with anti‐CD36 (AB17044; Lucerna‐Chem‐AG, Luzern, Switzerland) blocking antibody (1 μg/mL for 30 minutes at room temperature), which specifically blocks an epitope in CD36 receptor and a respective isotype control before exposure with oxidized albumin fractions (HMA [humanmercaptalbumin], HNA1, and HNA2, all at 1 mg/mL) to healthy platelets (Supporting Methods).

Techniques: Activation Assay, Incubation, Ex Vivo, Blocking Assay, Expressing

Journal: Hepatology Communications

Article Title: Hyperoxidized Albumin Modulates Platelets and Promotes Inflammation Through CD36 Receptor in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1440

Figure Lengend Snippet: Secretome analysis of platelets with CD36 blockade. (A) Experimental flow diagram of secretome analysis using high‐resolution mass spectrometry. (B) Heat map representation of secretome for HMA+CD36:HMA; HNA+CD36:HNA1; and HNA2+CD36:HNA2 represents distinct secretome in HNA2 group comparison. Red Venn represents proteins that are induced/increased, and green Venn represents proteins that get reduced/inhibited with CD36 neutralization ( P < 0.05). (C) Pathway overrepresentation analysis (Kyoto Encyclopedia of Genes and Genomes/Reactome) of HNA2‐specific 153 protein‐induced and 23 HNA2‐specific proteins inhibited under CD36 neutralization ( P < 0.05, C‐score > 1).

Article Snippet: Platelets were pretreated with anti‐CD36 (AB17044; Lucerna‐Chem‐AG, Luzern, Switzerland) blocking antibody (1 μg/mL for 30 minutes at room temperature), which specifically blocks an epitope in CD36 receptor and a respective isotype control before exposure with oxidized albumin fractions (HMA [humanmercaptalbumin], HNA1, and HNA2, all at 1 mg/mL) to healthy platelets (Supporting Methods).

Techniques: Mass Spectrometry, Neutralization

Journal: Hepatology Communications

Article Title: Hyperoxidized Albumin Modulates Platelets and Promotes Inflammation Through CD36 Receptor in Severe Alcoholic Hepatitis

doi: 10.1002/hep4.1440

Figure Lengend Snippet: Paradigm of platelet activation in SAH. Proposed proinflammatory/prothrombotic activities of oxidized albumin and CD36 receptor. In SAH platelets, HNA2 promotes inflammatory and oxidative activation through CD36 receptor–mediated autocrine signaling much more than HNA1. In brief, HNA2 interacts with CD36 and harbors the SNARE complex proteins, which are then used quickly for autocrine activation and reloading the membrane CD36 levels. These phenomena result in intracellular calcium mobilization, ROS production, and CD40L secretion. By blocking CD36, HNA2‐mediated activation of platelet through CD36 is attenuated and the SNARE complex proteins are available for normal degranulation of repair and regenerative proteins.

Article Snippet: Platelets were pretreated with anti‐CD36 (AB17044; Lucerna‐Chem‐AG, Luzern, Switzerland) blocking antibody (1 μg/mL for 30 minutes at room temperature), which specifically blocks an epitope in CD36 receptor and a respective isotype control before exposure with oxidized albumin fractions (HMA [humanmercaptalbumin], HNA1, and HNA2, all at 1 mg/mL) to healthy platelets (Supporting Methods).

Techniques: Activation Assay, Blocking Assay