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Blood, Vol. 110, Issue 2, 519-528, July 15, 2007
Mechanisms of induction of endothelial cell E-selectin expression by smooth muscle cells and its inhibition by shear stress
Blood Chiu et al.
110: 519
Supplemental materials for Chiu et al, Vol 110, Issue 2, 519-528
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 125 KB)
- Table S1. The expression levels of proteins identified by protein arrays (PDF, 288 KB) -
Protein array assays were used to measure the expressions of cytokines and other proteins in conditioned media of EC/EC and EC/SMC, as described in Document 1. Data are presented as the ratio of expression level of proteins in conditioned media of EC/SMC to that of EC/EC and are mean (± SEM) from four independent experiments for each protein. *Mean protein ratio ≥2 or ≤0.5 and P≤0.05 were considered statistically significant.
- Figure S1. Prolonged effects of high and low shear stresses on the activations of ERK, JNK, p38, Akt, and NF-κB, and the expression of E-selectin in ECs (JPG, 69.7 KB)
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ECs were kept as controls (CL) or exposed to a high shear stress of 12 dynes/cm2 (HS24) or a low shear stress of 0.5 dynes/cm2 (LS24) for 24 h. The cells were lysed and (A) the expression of E-selectin mRNA, the phosphorylation of (B) ERK, (C) JNK, (D) p38, and (E) Akt, and (F) the NF- B—DNA binding activity were determined by using RT-PCR, Western blot, and EMSA, respectively. The results are representative of three independent experiments with similar results.
- Figure S2. A template of the human cytokines and other proteins in the array (JPG, 149 KB)
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Pos: positive; Blank: negative. The full names of all abbreviations of the proteins in the array are provided in Table S1.
- Figure S3. Effects of neutralizing antibodies against various cytokines and chemokines on the co-culture-induced E-selectin expression in ECs (JPG, 35.7 KB)
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ECs were kept as controls (EC/Ø) or co-cultured with SMCs in the adjacent-bilayer model (EC/SMC) for 4 hours. Before co-culture with SMCs, ECs were incubated with a neutralizing antibody against bFGF (5 µg/mL), MCP-1 (10 µg/mL), GRO (10 µg/mL), RANTES (10 µg/mL), SDF-1 (30 µg/mL), I-TAC (5 µg/mL), IL-4 (5 µg/mL), or IL-1ra (20 µg/mL) for 1 hour. Control ECs were co-cultured with SMCs in the presence of control IgG (CL). The E-selectin mRNA expression was determined by RT-PCR. Incubation of ECs with IL-1ra, an IL-1 receptor antagonist, inhibited the co-culture-induced E-selectin expression, whereas incubation with other neutralizing antibodies did not have inhibitory effects on co-culture-induced E-selectin expression. Results are representative of triplicate experiments with similar results.
- Figure S4. Analysis of the expression and release of IL-6R by ECs and SMCs by RT-PCR and ELISA (JPG, 41.1 KB)
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(A) ECs were kept as controls (EC/Ø) or co-cultured with SMCs in an adjacent bilayer model (EC/SMC) for 1, 2, 4, and 24 hours. In parallel experiments, monocultures of SMCs were prepared but the opposite side had no cells (Ø/SMC). The cells were lysed and the expression of IL-6R mRNA was determined by using RT-PCR analysis (sense primer: 5′-CAAGCCTCCCAGTGCAAGAT-3′; antisense primer: 5′-ATTGCTGATGTCATAAGGGC-3′). The results are representative of three independent experiments with similar results. (B) The soluble forms of IL-6R (sIL-6R) in the media of EC and SMC monocultures were determined by ELISA (human sIL-6R kit, Catalog Number: DY227, R&D systems, Minneapolis, MN). The dishes incubated with only media were used as negative controls (NC). The results shown are mean (± SEM) from 3 independent experiments. *P<0.05 vs. NC, #P<0.05 vs. EC 1 day.
- Figure S5. Co-culture of ECs with SMCs induces associations of JNK and p38 with c-JUN and ATF-2, respectively (JPG, 43.2 KB)
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ECs kept as controls (EC/Ø) or co-cultured with SMCs in the adjacent-bilayer model (EC/SMC) for 30 minutes were collected by scraping and lysis. The extracts were immunoprecipitated with antibodies against JNK and p38, followed by Western blot analysis with antibodies against c-JUN (sc-1694, Santa Cruz Biotechnology) and ATF-2 (sc-187, Santa Cruz Biotechnology). The results show that co-culture of ECs with SMCs for 30 minutes induced the increases in JNK/c-JUN and p38/ATF-2 associations in ECs. ECs stimulated with TNF- (100 U/mL, 30 minutes) were used as positive controls to show increases in JNK/c-JUN and p38/ATP-2 associations.
- Figure S6. αvβ3 and β1 integrins and Src were not involved in SMC-co-culture and shear stress-modulations of E-selectin expression in ECs (JPG, 47.5 KB)
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(A) ECs were kept as controls (EC/Ø) or co-cultured with SMCs in the adjacent-bilayer model (EC/SMC) for 4 h. (B) In parallel experiments, ECs were pre-incubated with antibodies (10 µg/mL) against v3 and 1 integrins (MAB1976 and MAB 2253, respectively, Chemicon, Temecula, CA) or control IgG for 2 hours prior to seeding onto the membranes, and then subjected to a shear stress of 12 dynes/cm2 for 24 h (HS24) and/or co-cultured with SMCs in the presence of the respective antibodies. In some experiments, ECs were pre-treated with PP1 (10 µM, Sigma), a specific inhibitor of Src, for 30 minutes before and during the shear stress and/or co-culture experiments. The cells were lysed and the E-selectin mRNA expression was determined by using RT-PCR analysis. The results are representative of three independent experiments with similar results.
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