Figure 1.
Parallel plate culture system.
Endothelial cell monolayers were grown to confluence on glass coverslips, then affixed to parallel plate flow chambers (A) using a vacuum pump. Peristaltic pumps (B) are used to impose pulsatile flow of culture media (C) over the endothelial monolayers. The rotational speed of the pumps is controlled by an external computer (D) via RS-232 linkage.
Figure 2.
Physiologically modeled perfusion culture.
A 12–hour perfusion regime (hollow line) was developed by programming a series of real-time recordings in heart rate obtained in a healthy male subject into peristaltic pumps as ramp changes. For comparison, the average shear stress magnitude calculated over the entire 12–hour cycle (10.3 dynes/cm2 at 80 pulses/min) was programmed as steady pulsatile flow (dark line). Mean wall shear stress and pulse frequency experienced by the endothelial cells within parallel plate flow chamber over time are shown on the left and right axes, respectively. Insets show pressure waveforms experienced at various time points during steady (dark border) or physiological flow (hollow border).
Figure 3.
Cytoskeletal morphology of flow-conditioned endothelial cells.
Endothelial cell monolayers were grown to confluence and cultured under static conditions (A), steady flow (B), or physiological flow (C) for 24 hours. Monolayers were subsequently fixed and co-stained with rhodamine phalloidin (middle row) and DAPI (top row) in order to visualize f-actin and cell nuclei, respectively. Shown are representative images (40x) from each condition. Applying shear stress resulted in cytoskeletal alignment of endothelial cells in the flow direction (horizontally left to right). Scale bar: 20 microns.
Figure 4.
Cardio-protective gene expression in flow-conditioned endothelial cells.
Shown is fold mRNA expression (with respect to static-cultured EC) of genes that promote or inhibit cardiovascular disease progression. Results are presented as mean±SEM. An embedded asterisk indicates a significant difference with respect to static controls; an asterisk over a bracket indicates a significant difference between flow groups. Abbreviations: EDN1: endothelin-1; PTGIS: prostacyclin synthase; SOD1: superoxide dismutase-1.
Figure 5.
Coagulation & fibrinolysis-associated gene expression in flow-conditioned endothelial cells.
Shown is fold mRNA expression (with respect to static-cultured EC) of genes associated with hemostasis (A) and fibrinolysis (B). Results are presented as mean±SEM. An embedded asterisk indicates a significant difference with respect to static controls; an asterisk over a bracket indicates a significant difference between flow groups. Abbreviations: ANXA5: annexin V; TFPI: tissue factor pathway inhibitor; THBD: thrombomodulin; PLAT: tissue plasminogen activator; PLAU: urokinase plasminogen activator; PAI-1: plasminogen activator inhibitor-1.
Figure 6.
Inflammation-associated gene expression in flow-conditioned endothelial cells.
Shown is fold mRNA expression (with respect to static-cultured endothelial cells) of cell adhesion molecules (A) and genes with roles in recruitment of inflammatory cells (B). Results are presented as mean±SEM. An embedded asterisk indicates a significant difference with respect to static controls; an asterisk over a bracket indicates a significant difference between flow groups. Abbreviations: PSGL-1: P-selectin glycoprotein ligand-1; ICAM-1: intercellular adhesion molecule-1; VCAM-1: vascular cell adhesion molecule-1; PECAM-1: platelet-endothelial cell adhesion molecule-1; ALOX5: arachidonate 5-lipoxygenase; PLA2G4C: cytosolic phospholipase A2 gamma; ADAM17: ADAM metallopeptidase domain 17; MCP-1: monocyte chemoattractant protein-1; CX3CL1: fractalkine.
Figure 7.
eNOS function in flow-conditioned endothelial cells.
(A): eNOS mRNA expression (presented as mean±S.E.M.) was upregulated under either perfusion condition, but no statistical difference between flow groups was observed. (B): After 0, 12, or 24 hours of conditioning, media was collected and samples analyzed using a fluorometric assay. Total NO byproduct accumulation was normalized by the mean cell count at the end of each period. Results are displayed as mean±SEM (n = 4–6). Asterisks denote significant differences in individual means between groups at each time point. Dagger (†) denotes a significant difference in mean with respect to t = 0 hours. Double dagger (‡) denotes significance with respect to t = 0 and t = 12 hours.
Figure 8.
HL-60 cell adhesion to flow-conditioned endothelial cells.
Endothelial cell monolayers were grown to confluence and cultured under steady flow (A,C,E,G) or physiological flow (B,D,F,H) for 24 hours. During the last four hours, endothelial cells were activated with 1 U TNF-a to stimulate adhesion molecule expression. At hour 24, monolayers were removed from flow chambers and incubated for 10 minutes with a bolus of GFP+ HL-60 cells (1000 cells/mm2) and stained as described (A,B: DAPI; C,D: F-actin; E,F: GFP+ HL-60 cells; G,H: overlay). Shown are representative images (40x) from each condition. Scale bar: 20 microns. (I): HL-60 cell adhesion in 15 predetermined locations per monolayer was quantified. Results are displayed as mean±SEM (n = 5–6). Asterisk denotes significant difference in means between groups as determined by Student’s t-test.