Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Platelet CD40 Exacerbates Atherosclerosis by Transcellular Activation of Endothelial Cells and Leukocytes

doi: 10.1161/atvbaha.115.307074

Figure Lengend Snippet: Figure 4. Platelet cluster of differentiation 40 (CD40) deficiency reduces endothelial activation and neutrophil recruitment. A and B, Intra- vital microscopic analysis of platelet adhesion to endothelium in the carotid artery of Apoe−/− mice treated with tumor necrosis factor (TNF)-α (500 ng, IP) for 30 min and subsequently infused with thrombin-activated platelets (0.5 nmol/L, 15 min; 3×107/20 g) isolated from Apoe−/− or Cd40−/−Apoe−/− mice (n=4). C–H, Intravital microscopy of carotid arteries from Apoe−/− mice fed a Western-type diet for 4 wk and injected with Apoe−/− or Cd40−/−Apoe−/− platelets every fifth day. C and D, Interactions of leukocytes with the carotid artery visualized by rhodamine injection. E and F, Inflammatory monocytes and neutrophils were visualized with an antibody to Gr1. G and H, Luminal arrest of fluorescent beads conjugated with antibody to vascular cell adhesion molecule (VCAM)-1 (n=6; scale bar, 100 µm for B, D, F, and H). I, Quantitative polymerase chain reaction to determine mRNA expression of various adhesion molecules and inflammatory media- tors in endothelial cells (SVEC4-10) coincubated for 12 h with thrombin-activated platelets from Apoe−/− or Cd40−/−Apoe−/− mice. Data are expressed relative to the control group (n=4). J, Confluent endothelial cells (SVEC4-10), stimulated with TNF-α (10 ng/mL; 4 h) were exposed in flow chambers to perfusion with thrombin-activated platelets from Apoe−/− or Cd40−/−Apoe−/− mice for 20 min followed by per- fusion with calcein-labeled leukocytes (RAW 264.7). Adherent cells per field of view were counted. Similar leukocyte adhesion flow cham- ber assays performed in the presence of (K) anti-VCAM and (L) anti–IL-1β antibodies or the respective isotype-matched controls (n=4, 8 replicate fields; for I–K). *P<0.05. ICAM indicates intercellular adhesion molecule; and PECAM, platelet endothelial cell adhesion molecule.

Article Snippet: SV40-immortalised mouse endothelial cells (SVEC4-10 (ATCC® CRL-2181TM)) were challenged with tumor necrosis factor-α (10 ng/mL; Peprotec) for 4 hours.

Techniques: Activation Assay, Isolation, Intravital Microscopy, Western Blot, Injection, Real-time Polymerase Chain Reaction, Expressing, Control, Labeling

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137L promotes the proliferation, migration and tube formation of lymphatic endothelial cells. (A) The proliferation of LECs treated with CD137L or CD137L and inhibitory for 24 h, 48 h, and 72 h was examined by CCK-8 assay. The migration of LECs was measured with the cell scratch test (B) and Transwell migration assay (D) after CD137L or CD137L and inhibitory treatment for 12 h. The histograms represent migrated (C, E) cells per field. (F) A tube formation assay was used to test the lymphangiogenesis capacity of LECs after CD137L or CD137L and inhibitory treatment for 3 h (image magnification, 100×). The histograms represent the branch number per field. (G, H, I). The error bars represent the SEM. *P< 0.05, **P < 0.01; ***P < 0.001. The data were pooled from three independent experiments.

Article Snippet: The mouse lymph node endothelial cell line (SVEC4-10, LECS) was purchased from the China Center for Type Culture Collection (CCTCC).

Techniques: Migration, CCK-8 Assay, Transwell Migration Assay, Tube Formation Assay

Journal: International Journal of Biological Sciences

Article Title: CD137L-macrophage induce lymphatic endothelial cells autophagy to promote lymphangiogenesis in renal fibrosis

doi: 10.7150/ijbs.66781

Figure Lengend Snippet: CD137L induced autophagy of lymphatic endothelial cells via mTOR pathway. (A) Representative electron micrographs of CD137L-induced autophagosome in SVEC4-10 cells that were either stimulated with inhibitory, or with 3-MA for 24 h. (B) The protein expression levels of autophagy-associated proteins (Atg5, Atg7 and Atg12) and autophagic substrates (LC3 and p62) were analyzed in SVEC4-10 cells after CD137L, inhibitory or 3-MA treatment for 24 h by Western blot. (C) SVEC4-10 cells were infected with RFP-GFP-LC3-expressing lentivirus. Cells were untreated or treated with CD137L, inhibitory or 3-MA for 24 h. Fluorescence was examined by confocal microscopy. Transwell migration assay (D), scratch test (E) and tube formation assay (F) stimulated with CD137L for 12 h, and with or without 3-MA treatment. (G) The protein expression levels of phosphorylated PI3K, Akt and mTOR were analyzed in SVEC4-10 cells after CD137L or inhibitory or 3-MA treatment for 24 h by Western blot. (H) SVEC4-10 cells were knocked down Atg5 and Atg7 by using siRNAs, and stimulated with CD137L for 24h, the protein expression levels of autophagy-associated proteins (Atg5, Atg7 and Atg12) and autophagic substrates (LC3 and p62) were detected by Western blotting.

Article Snippet: The mouse lymph node endothelial cell line (SVEC4-10, LECS) was purchased from the China Center for Type Culture Collection (CCTCC).

Techniques: Expressing, Western Blot, Infection, Fluorescence, Confocal Microscopy, Transwell Migration Assay, Tube Formation Assay