Figure 3
Figure 3. Induction of EC E-selectin expression by SMC-coculture and its inhibition by shear stress are mediated by JNK and p38 pathways. ECs were kept as controls (EC/∅) or cocultured with SMCs in the adjacent-bilayer model (EC/SMC) for 30 minutes (C,E), 4 hours (A,D), or 24 hours (B,F). Before kept as controls or coculture with SMCs, ECs were (1) pretreated with PD98059 (PD; 30 μM), SP600125 (SP; 20 μM), SB203580 (SB; 10 μM), or LY294002 (LY; 30 μM) individually or SP600125 and SB203580 simultaneously (CB) for 1 hour (A-B); (2) presheared at HSS (HS) or LSS (LS) for 24 hours (C); or (3) transfected with control siRNA or a specific siRNA of ERK, JNK, p38, or Akt (100 μmol/mL for each), or empty vector control PSRα (1 μg/mL), RasN17 (1 μg/mL), RacN17 (0.5 μg/mL), JNK(K-R) (1 μg/mL), Raf301 (1 μg/mL), or mERK (0.25 μg/mL) for 48 hours (D-F). Control ECs (CL) were cocultured with SMCs without any pretreatment (A) or preshearing (C). In some experiments, ECs were transfected with different siRNA and chimera at various concentrations for 48 hours (E). (F) ECs were cotransfected with chimera (2 μg) containing 540 bp (base pair) of E-selectin promoter region and the reporter gene luciferase. The mRNA (A,D) or surface protein (B) expression or promoter activity (F) of E-selectin or the expression or phosphorylation (C,E) of different MAPKs or Akt in these ECs was determined by using Northern blot (A), flow cytometric (B), Western blot (C,E), real-time PCR (D), or luciferase assay analysis (F), respectively, as described in “Materials and methods.” The results are shown as mean ± SEM from 3 or 4 separate experiments (A,C,D,F) or are representative of triplicate experiments with similar results (B,E). Data are presented as percentage changes in band density from control EC/∅ (A,C); normalized to 18S RNA (A) or JNK or p38 protein level (C). (B) ECs incubated with FITC-conjugated control IgG or FITC-conjugated antibody alone were used as IgG controls or negative controls (ie, Blanks: B). Numbers are mean ± SEM of mean fluorescent intensity for all experiments determined by comparison with corresponding negative controls. *P < .05 versus control EC/∅. #P < .05 versus control EC/SMC. $P < .05 versus cells transfected with control siRNA or empty vector control PSRα.

Induction of EC E-selectin expression by SMC-coculture and its inhibition by shear stress are mediated by JNK and p38 pathways. ECs were kept as controls (EC/∅) or cocultured with SMCs in the adjacent-bilayer model (EC/SMC) for 30 minutes (C,E), 4 hours (A,D), or 24 hours (B,F). Before kept as controls or coculture with SMCs, ECs were (1) pretreated with PD98059 (PD; 30 μM), SP600125 (SP; 20 μM), SB203580 (SB; 10 μM), or LY294002 (LY; 30 μM) individually or SP600125 and SB203580 simultaneously (CB) for 1 hour (A-B); (2) presheared at HSS (HS) or LSS (LS) for 24 hours (C); or (3) transfected with control siRNA or a specific siRNA of ERK, JNK, p38, or Akt (100 μmol/mL for each), or empty vector control PSRα (1 μg/mL), RasN17 (1 μg/mL), RacN17 (0.5 μg/mL), JNK(K-R) (1 μg/mL), Raf301 (1 μg/mL), or mERK (0.25 μg/mL) for 48 hours (D-F). Control ECs (CL) were cocultured with SMCs without any pretreatment (A) or preshearing (C). In some experiments, ECs were transfected with different siRNA and chimera at various concentrations for 48 hours (E). (F) ECs were cotransfected with chimera (2 μg) containing 540 bp (base pair) of E-selectin promoter region and the reporter gene luciferase. The mRNA (A,D) or surface protein (B) expression or promoter activity (F) of E-selectin or the expression or phosphorylation (C,E) of different MAPKs or Akt in these ECs was determined by using Northern blot (A), flow cytometric (B), Western blot (C,E), real-time PCR (D), or luciferase assay analysis (F), respectively, as described in “Materials and methods.” The results are shown as mean ± SEM from 3 or 4 separate experiments (A,C,D,F) or are representative of triplicate experiments with similar results (B,E). Data are presented as percentage changes in band density from control EC/∅ (A,C); normalized to 18S RNA (A) or JNK or p38 protein level (C). (B) ECs incubated with FITC-conjugated control IgG or FITC-conjugated antibody alone were used as IgG controls or negative controls (ie, Blanks: B). Numbers are mean ± SEM of mean fluorescent intensity for all experiments determined by comparison with corresponding negative controls. *P < .05 versus control EC/∅. #P < .05 versus control EC/SMC. $P < .05 versus cells transfected with control siRNA or empty vector control PSRα.

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