Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Synergistic effect of thrombin and CD40 ligand on endothelial matrix metalloproteinase-10 expression and microparticle generation in vitro and in vivo.

doi: 10.1161/ATVBAHA.112.248773

Figure Lengend Snippet: Figure 1. Synergistic effect of thrombin/CD40 ligand (CD40L) on human endothelial cells (ECs). A, Matrix metalloproteinase-10 (MMP-10) mRNA (black) and protein levels (white) in human umbilical vein endothelial cells upon increasing concentration of CD40L (0.1–1 g/mL). B, Time course of endothelial MMP-10 expression (meanSD, n4) after stimulation with thrombin (0.25 U/mL; squares), CD40L (0.25 g/mL; triangles), or throm- bin/CD40L (circles). C, MMP-10 mRNA levels in human aortic endothelial cells 12 hours after stimulation (as in B). D, Thrombin (0.25 U/mL) and CD40L (0.25 g/mL) increased MMP-10 mRNA (black) and protein (white) levels in ECs 12 hours after stimula- tion (meanSEM, n6). A protease-activated receptor-1 (PAR-1) agonist peptide (thrombin receptor agonist peptide [TRAP]-1; 50 M) mimicked the effect of thrombin on MMP-10 expression, whereas no effect was observed for a PAR-2 agonist (TRAP-2; 100 M) (E and F). Thrombin/CD40L-induced (12 hours) endothelial MMP-10 upregulation (mRNA [E] and protein [F]) was prevented by blocking CD40L (-CD40L; 7.5 µg/mL), throm- bin (hirudin; 10 M), -PAR-1 (ATAP-2; 1.2 g/mL), or CD40 (-CD40; 7.5 g/mL). No effect was observed for -PAR-1 (N-19; 1.2 g/mL) or isotype control antibodies (7.5 g/mL) (*P0.05; **P0.01; ***P0.001 vs control, and †P<0.05 and ‡P<0.01 vs thrombin/CD40L).

Article Snippet: Blots were incubated overnight with a monoclonal antibody -CD40 (2 μg/mL, R&D Systems) followed by incubation with a peroxidase-conjugated rabbit anti-mouse antibody (GE Healthcare) and developed by a chemiluminescent detection system (ECL Advance, GE Healthcare).

Techniques: Concentration Assay, Expressing, Blocking Assay, Control

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Synergistic effect of thrombin and CD40 ligand on endothelial matrix metalloproteinase-10 expression and microparticle generation in vitro and in vivo.

doi: 10.1161/ATVBAHA.112.248773

Figure Lengend Snippet: Figure 2. Induction of matrix metalloproteinase-10 (MMP-10) by CD40 ligand (CD40L) and thrombin/CD40L via mitogen-activated protein kinase (MAPK). A, Western blot analysis showing the time-dependent increase in the phosphorylation of extracellular signaling-regulated kinases (ERK)-1/2 (p-ERK), p38 MAPK (p-p38), and c-Jun N-terminal kinase-1 (JNK-1; p-JNK) in human umbilical vein endothelial cells (HUVEC) treated with thrombin (0.25 U/mL; upper panel), CD40L (0.25 g/mL; middle panel), or thrombin/CD40L (lower panel). B, C, MMP-10 mRNA levels in CD40L (B) and thrombin/CD40L (C) stimulated HUVEC in presence of different inhibitors: 10 M PD98059 (ERK1/2 inhibitor), 10 M SB203580 (p38 MAPK inhibitor), and 10 µM SP600125 (JNK-1 inhibitor). Inhibition of p38 MAPK and JNK-1 signaling pathways significantly prevented MMP-10 upregulation. D and E, MMP-10 protein levels (ELISA) in CD40L (D) and throm- bin/CD40L (E) stimulated HUVEC in the presence of different inhibitors (as in B and C: (n4, *P<0.05, **P0.01 vs CD40L or CD40L/ thrombin).

Article Snippet: Blots were incubated overnight with a monoclonal antibody -CD40 (2 μg/mL, R&D Systems) followed by incubation with a peroxidase-conjugated rabbit anti-mouse antibody (GE Healthcare) and developed by a chemiluminescent detection system (ECL Advance, GE Healthcare).

Techniques: Western Blot, Phospho-proteomics, Inhibition, Protein-Protein interactions, Enzyme-linked Immunosorbent Assay

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Synergistic effect of thrombin and CD40 ligand on endothelial matrix metalloproteinase-10 expression and microparticle generation in vitro and in vivo.

doi: 10.1161/ATVBAHA.112.248773

Figure Lengend Snippet: Figure 3. Thrombin increases CD40 expression in endothelial cells (ECs). A, CD40 mRNA levels in human umbilical vein endothelial cells (HUVEC) stimulated for 12 hours with thrombin (0.5 U/mL), CD40L (0.5 g/mL), thrombin receptor agonist peptide (TRAP)-1 (50 M), and thrombin coincubated with CD40 ligand (CD40L), hirudin (10 M), ATAP-2, or N-19 (1.2 µg/mL) (meanSD, n4). B, Western blot analysis of CD40 receptor (48 kDa) in lysates from HUVEC stimulated with thrombin (24 hours). Histogram represents CD40/-actin ratio. C, Flow cytometry analysis of CD40 ECs stimulated with thrombin for 24 hours (dashed pink line) compared with controls (solid green line). D, Immunofluorescence for CD40 (green) in HUVEC treated with thrombin (30 minutes, 0.25 U/mL). Insets show high-power magnification. Draq5 (blue) was used as nuclear staining (*P0.05 vs controls and ‡P0.01 vs thrombin).

Article Snippet: Blots were incubated overnight with a monoclonal antibody -CD40 (2 μg/mL, R&D Systems) followed by incubation with a peroxidase-conjugated rabbit anti-mouse antibody (GE Healthcare) and developed by a chemiluminescent detection system (ECL Advance, GE Healthcare).

Techniques: Expressing, Western Blot, Flow Cytometry, Immunofluorescence, Staining

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Synergistic effect of thrombin and CD40 ligand on endothelial matrix metalloproteinase-10 expression and microparticle generation in vitro and in vivo.

doi: 10.1161/ATVBAHA.112.248773

Figure Lengend Snippet: Figure 4. Pathophysiological role of matrix metalloproteinase-10 (MMP-10) in vivo. A, MMP-10 protein levels (Western blot) were upregu- lated in aortic lysates from mice 24 hours after thrombin (2.5 U) or CD40 ligand (CD40L; 2.5 µg) administration, although the strongest upregulation was achieved by the combination of both stimuli. rhMMP-10 (proenzyme: 55 kDa; active enzyme: 45 kDa) was used as posi- tive control and -actin (42 kDa) as loading control (n14, *P0.05 vs controls). Results are expressed as the ratio between MMP-10 and -actin and expressed as fold increase relative to controls. B, Morphological analysis for MMP-10 in mouse aortas after thrombin/CD40L administration. C, Circulating levels of MMP-10, CD40L, and D-dimer (meanSEM) in patients with sepsis (black bars; n=60) and controls (gray bars; n50). D, Survival curve in lipopolysaccharide-injected (5 mg/kg) wild-type and Mmp10 mice (n=20/genotype) (*P0.05, ***P0.001).

Article Snippet: Blots were incubated overnight with a monoclonal antibody -CD40 (2 μg/mL, R&D Systems) followed by incubation with a peroxidase-conjugated rabbit anti-mouse antibody (GE Healthcare) and developed by a chemiluminescent detection system (ECL Advance, GE Healthcare).

Techniques: In Vivo, Western Blot, Control, Injection

Journal: Arteriosclerosis, thrombosis, and vascular biology

Article Title: Synergistic effect of thrombin and CD40 ligand on endothelial matrix metalloproteinase-10 expression and microparticle generation in vitro and in vivo.

doi: 10.1161/ATVBAHA.112.248773

Figure Lengend Snippet: Figure 5. Generation of microparticles (MPs) in vivo and in vitro. A, Detection (flow cytometry) of circulating MPs in saline or lipoplysac- charide (LPS)-injected (15 mg/kg, n3/condition) wild-type (WT) and Mmp10 mice. B, Determination of cellular origin (flow cytom- etry) of circulating MPs in control and LPS-injected WT and Mmp10 mice; CD45 MPs are leukocyte-derived vesicles, CD31 MPs correspond to endothelial, platelet, or leukocyte origin, and CD41 MPs are platelet-derived vesicles (n3/condition). C, Circulating CD41 MPs were immunostained for CD40 ligand (CD40L) in saline and LPS-injected WT and Mmp10 mice (n3condition). D, MPs from different cellular origins harbor MMP-10 in WT mice (n3/condition). E, Western blot for matrix metalloproteinase-10 (MMP-10) in human umbilical vein endothelial cell (HUVEC) supernatants 24 hours after stimulation with thrombin (0.25 U/mL), CD40L (0.25 gmL), or the combination of both of them before and after MP removal. Active MMP-10 was detected (45 kDa). F, lower panel: quantification of endothelial-derived MPs (EMPs) originated from HUVEC stimulated with thrombin (1 U/mL), CD40L (0.25 g/mL), or thrombinCD40L for 48 hours. Upper panel: MMP-10 (Western blot) in EMPs originated from cells stimulated with thrombin (Thr, 10 U/mL) CD40L (1 µg/mL). G, CD40L (18 kDa) was detected by Western blot in lysed platelet-derived MPs (PMPs; upper panel). rhCD40L was used as positive control (0.25 g/mL). Lower panel: Endothelial MMP-10 expression 6 hours after stimulation with thrombin (0.25 U/mL), CD40L PMPs (0.6 mg/mL), and CD40L PMPs preincubated with -CD40L antibody (7.5 µg/mL) before thrombin addition. Data (meanSD, n3) (*P<0.05 vs controls, **P0.01 vs thrombin, †P0.05 vs thrombin/PMPs).

Article Snippet: Blots were incubated overnight with a monoclonal antibody -CD40 (2 μg/mL, R&D Systems) followed by incubation with a peroxidase-conjugated rabbit anti-mouse antibody (GE Healthcare) and developed by a chemiluminescent detection system (ECL Advance, GE Healthcare).

Techniques: In Vivo, In Vitro, Flow Cytometry, Saline, Injection, Control, Derivative Assay, Western Blot, Positive Control, Expressing