1. Exposure to fluid shear stress modulates the ability of endothelial cells to recruit neutrophils in response to tumor necrosis factor-α: a basis for local variations in vascular sensitivity to inflammation
by Sajila Sheikh, G. Ed Rainger, Zoe Gale, Mahbub Rahman, and Gerard B. Nash
Blood
Volume 102(8):
October 15, 2003
©2003 by American Society of Hematology

2. Schematic diagrams of flow culture system and flow-based adhesion assay.
Schematic diagrams of flow culture system and flow-based adhesion assay. (A) Flow culture system. Medium in the culture dish was constantly perfused at a chosen shear stress through the microslide containing HUVEC-1 and perfused for 30 seconds each hour at low shear stress through the microslide containing HUVEC-2. The 2 microslides received identical medium. (B) Flow-based adhesion assay. Either neutrophil suspension or wash buffer was perfused at a wall shear stress of 0.1 Pa through the microslide. HUVECs and neutrophils adhering to it were continually observed and recorded by phase-contrast videomicroscopy.
Sajila Sheikh et al. Blood 2003;102:
©2003 by American Society of Hematology

3. Effect of exposing HUVECs to flow on their response to different concentrations of TNF. Response was assessed by (A) number of adherent neutrophils, and (B) percentage of adherent neutrophils transmigrating through the monolayer.
Effect of exposing HUVECs to flow on their response to different concentrations of TNF. Response was assessed by (A) number of adherent neutrophils, and (B) percentage of adherent neutrophils transmigrating through the monolayer. HUVECs were cultured static (□) or exposed to a shear stress of 0.3 Pa (▨) for 28 hours, with TNF added for the last 4 hours, followed by flow-based adhesion assay. Data are mean ± SEM from 3 or 4 experiments at each concentration of TNF. Analysis of variance (ANOVA) showed significant effect of culture conditions on adhesion (P < .05) and transmigration (P < .01). *P < .05, **P < .01 compared with results for static cultures by paired t test.
Sajila Sheikh et al. Blood 2003;102:
©2003 by American Society of Hematology

4. Effect of exposing HUVECs to flow for varying times on their response to TNF. Response was assessed by the percentage of adherent neutrophils transmigrating through the monolayer.
Effect of exposing HUVECs to flow for varying times on their response to TNF. Response was assessed by the percentage of adherent neutrophils transmigrating through the monolayer. HUVECs were cultured static (□) or exposed to shear stress of 0.3 Pa (▨) for 4, 8, or 28 hours. TNF (100 U/mL) was added for the last 4 hours, followed by flow-based adhesion assay. Data are mean ± SEM from 3 or 4 experiments of each duration. ANOVA showed significant effect of culture conditions on transmigration (P < .01). *P < .05, **P < .01 compared with results for static cultures by paired t test.
Sajila Sheikh et al. Blood 2003;102:
©2003 by American Society of Hematology

5. Effect of exposing HUVECs to flow at different shear stresses on their response to TNF. Response was assessed by the number of adherent neutrophils.
Effect of exposing HUVECs to flow at different shear stresses on their response to TNF. Response was assessed by the number of adherent neutrophils. HUVECs were cultured static (□) or exposed to flow (▨) at a shear stress of 0.3, 1.0, or 2.0 Pa for 28 hours with 100 U/mL TNF added for the last 4 hours, followed by flow-based adhesion assay. Data are mean ± SEM from 3 to 5 experiments at each stress. ANOVA showed significant effect of the level of stress on adhesion (P < .01). *P < .02 compared with results for static cultures by paired t test.
Sajila Sheikh et al. Blood 2003;102:
©2003 by American Society of Hematology

6. Effect of exposing HUVECs to flow on their expression of genes for chemokines and adhesion receptors in response to TNF. (A) Stained gels showing DNA amplified by RT-PCR from mRNA extracted from HUVECs that were cultured for 26 hours under static conditions...
Effect of exposing HUVECs to flow on their expression of genes for chemokines and adhesion receptors in response to TNF. (A) Stained gels showing DNA amplified by RT-PCR from mRNA extracted from HUVECs that were cultured for 26 hours under static conditions (S) or exposed to shear stress of 0.3 Pa or 2 Pa (F). TNF (100 U/mL) was added for the last 2 hours. Actin is shown as a loading control unmodified by treatment. Gels are from individual experiments, which were carried out on 3 occasions. (B) Densitometry of DNA bands obtained by RT-PCR for HUVECs treated with 100 U/mL TNF and cultured under static conditions or exposed to shear stress of 0.3 Pa or 2.0 Pa. Data are mean ± SEM from 3 experiments under each condition. ANOVA showed significant effect of culture conditions on expression of IL-8, GRO-α, and E-selectin (*P < .05, **P < .01).
Sajila Sheikh et al. Blood 2003;102:
©2003 by American Society of Hematology

7. Western blots from HUVECs cultured static or at 0. 3 or 2. 0 Pa
Western blots from HUVECs cultured static or at 0.3 or 2.0 Pa. (A) IL-8 and P-selectin for HUVECs that had been cultured for 24 hours static or under flow, and then a further 4 hours with 100 U/mL TNF. (B) TNF receptors 1 and 2 (p55TNFR and p75TNFR) for HUV...
Western blots from HUVECs cultured static or at 0.3 or 2.0 Pa. (A) IL-8 and P-selectin for HUVECs that had been cultured for 24 hours static or under flow, and then a further 4 hours with 100 U/mL TNF. (B) TNF receptors 1 and 2 (p55TNFR and p75TNFR) for HUVECs that had been cultured for 24 hours static or under flow. Actin is shown as a loading control in each case. Positions of bands for P-selectin and IL-8 were determined by comparisons to purified proteins run in separate lanes and to molecular weight standards. Bands for TNF receptors were consistent with known molecular weights. Western blots are from individual experiments, which were repeated on 2 to 4 occasions with similar results.
Sajila Sheikh et al. Blood 2003;102:
©2003 by American Society of Hematology

8. Effect of exposing HUVECs to flow on their up-regulation of surface expression of E-selectin in response to treatment with TNF. HUVECs were cultured under static conditions or exposed to shear stress of 0.3 Pa or 2.0 Pa for 28 hours, with 100 U/mL TNF added...
Effect of exposing HUVECs to flow on their up-regulation of surface expression of E-selectin in response to treatment with TNF. HUVECs were cultured under static conditions or exposed to shear stress of 0.3 Pa or 2.0 Pa for 28 hours, with 100 U/mL TNF added for the last 4 hours. Data are mean ± SEM from 3 experiments using immunofluorescence labeling and flow cytometry. Values are expressed relative to intensity of fluorescence for HUVECs labeled with nonspecific control antibody. *P < .05 compared with HUVECs treated with TNF under static conditions. Horizontal line represents relative fluorescence intensity (set at 1).
Sajila Sheikh et al. Blood 2003;102:
©2003 by American Society of Hematology